Unconventional nucleotide substitution in temperature selective RT-PCR

ABSTRACT

Methods are provided for distinguishing between RNA and DNA templates in an amplification reaction. In a preferred embodiment of the invention, the amplification reaction is a PCR and the reaction is catalyzed by a thermostable DNA polymerase or both reverse transcription and amplification of a target RNA. The invention particularly relates to selective amplification of RNA in the presence of homologous DNA, for example, HIV nucleic acids.

CROSS-REFERENCE

This application is a continuation of application Ser. No. 08/082,182,filed Jun. 24, 1993, which issued as U.S. Pat. No. 5,310,652, which is acontinuation of U.S. Ser. No. 07/746,121, filed Aug. 15, 1991,abandoned, which is a continuation-in-part (CIP) of copendingPCT/US90/07641, filed Dec. 21, 1990, which is a CIP of Ser. No. 585,471,filed Sep. 20, 1990, now abandoned, which is a CIP of Ser. No. 455,611,filed Dec. 22, 1989, which issued as U.S. Pat. No. 5,322,770. Thisapplication is also a CIP of Ser. No. 455,967, filed Dec. 22, 1989, nowabandoned, which is a CIP of Ser. No. 143,441, filed Jan. 12, 1988, nowabandoned, which is a CIP of Ser. No. 063,509, filed Jun. 17, 1987,which issued as U.S. Pat. No. 4,889,818, and which is a CIP of nowabandoned Ser. No. 899,241, filed Aug. 22, 1986. This application isalso a CIP of Ser. No. 609,157, filed Nov. 2, 1990, now abandoned whichis a CIP of Ser. No. 557,517, filed Jul. 24, 1990, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of molecular biology andprovides improved methods for the replication and amplification ofribonucleic acid (RNA) sequences. In a preferred embodiment, theinvention provides a method for synthesizing a complementary DNA copyfrom an RNA template with a thermoactive DNA polymerase. In anotheraspect, the invention provides methods for coupling reversetranscription of an RNA template and amplification of the resultant DNAusing a thermostable DNA polymerase. In a preferred embodiment RNA isreverse transcribed and amplified in a homogeneous, one tube, one enzymereaction. Methods for sterilization of reverse transcription and reversetranscription/amplification reactions are also provided.

2. Description of Related Art

The term "reverse transcriptase" describes a class of polymerasescharacterized as RNA-dependent DNA polymerases. All known reversetranscriptases require a primer to synthesize a DNA transcript from anRNA template. Historically, reverse transcriptase has been usedprimarily to transcribe mRNA into cDNA which can then be cloned into avector for further manipulation.

Avian myoblastosis virus (AMV) reverse transcriptase was the firstwidely used RNA-dependent DNA polymerase (Verma, 1977, Biochem. Biophys.Acta 473:1). The enzyme has 5'-3' RNA-directed DNA polymerase activity,5'-3' DNA-directed DNA polymerase activity, and RNase H activity. RNaseH is a processive 5' and 3' ribonuclease specific for the RNA strand ofRNA-DNA hybrids (Perbal, 1984, A Practical Guide to Molecular Cloning,Wiley & Sons New York). Errors in transcription cannot be corrected byreverse transcriptase because known viral reverse transcriptases lackthe 3'→5' exonuclease activity necessary for proofreading (Saunders andSaunders, 1987, Microbial Genetics Applied to Biotechnology, Croom Helm,London). A detailed study of the activity of AMV reverse transcriptaseand its associated RNase H activity has been presented by Berger et al.,1983, Biochemistry 22:2365-2372.

Berger et al. found that the rate limiting step in the reversetranscription of RNA was initiation by the enzyme, rather than thesequential polymerization of additional nucleotides. To overcome thislimitation, use of a stoichiometric, rather than catalytic, quantity ofreverse transcriptase is frequently recommended (Buell et al., 1978, J.Biol. Chem. 253:2471-2482; Wickens et al., 1978, J. Bio. Chem.253:2483-2495; Yoo et al., 1982, Proc. Nat. Acad. Sci. USA 80:1194-1198;and Okayama and Berg, 1982, Mol. Cell. Bio. 2:161-170). However, whenstoichiometric amounts of reverse transcriptase are used, the low levelof RNase H activity is significant and may be responsible for fragmentedcDNAs and limited cDNA yields (Kotewicz et al., 1988, Nuc. Acid Res.16:265-277). Christopher et al., 1980, Eur. J. Biochem. 111:4190-4231,and Michelson et al., 1983, Proc. Nat. Acad. Sci. USA 80:472-476, havesuggested that including an RNase inhibitor in cDNA reactions couldalleviate this problem.

DNA polymerases isolated from mesophilic microorganisms such as E. coli.have been extensively researched (see, for example, Bessman et al.,1957, J. Biol. Chem. 233:171-177 and Buttin and Kornberg, 1966, J. Biol.Chem. 241:5419-5427). E. Coli DNA polymerase I (Pol I) is useful for anumber of applications including: nick-translation reactions, DNAsequencing, in vitro mutagenesis, second strand cDNA synthesis,polymerase chain reactions (PCR), and blunt end formation for linkerligation (Maniatis et al., 1982, Molecular Cloning: A Laboratory ManualCold Spring Harbor, N.Y.).

Several laboratories have shown that some DNA polymerases are capable ofin vitro reverse transcription of RNA (Karkas, 1973, Proc. Nat. Acad.Sci. USA 70:3834-3838; Gulati et al, 1974, Proc. Nat. Acad. Sci. USA71:1035-1039; and Wittig and Wittig, 1978, Nuc. Acid Res. 5:1165-1178).Gulati et al. found that E. Coli Pol I could be used to transcribe Qβviral RNA using oligo(dT)₁₀ as a primer. Wittig and Wittig have shownthat E. coli Pol I can be used to reverse transcribe tRNA that has beenenzymatically elongated with oligo(dA). However, as Gulati et al.demonstrated, the amount of enzyme required and the small size of thecDNA product suggests that the reverse transcriptase activity of E. coliPol I has little practical value.

The use of thermostable enzymes to amplify existing nucleic acidsequences in amounts that are large compared to the amount initiallypresent was described in U.S. Pat. Nos. 4,683,195 and 4,683,202, whichdescribe the polymerase chain reaction (PCR) processes. These patentsare incorporated herein by reference. Primers, template, nucleosidetriphosphates, the appropriate buffer and reaction conditions, and apolymerase are used in the PCR process, which involves denaturation oftarget DNA, hybridization of primers, and synthesis of complementarystrands. The extension product of each primer becomes a template for theproduction of the desired nucleic acid sequence. These patents disclosethat, if the polymerase employed is a thermostable enzyme, thenpolymerase need not be added after every denaturation step, because heatwill not destroy the polymerase activity.

Thermostable DNA polymerases are not permanently inactivated even whenheated to 93°-95° C. for brief periods of time, as, for example, in thepractice of DNA amplification by PCR. In contrast, at this elevatedtemperature E. coli DNA Pol I and previously described reversetranscriptases are inactivated.

The thermostable DNA polymerase from Thermus aquaticus (Taq) has beencloned, expressed, and purified from recombinant cells as described inLawyer et al., 1989, J. Biol. Chem. 264:6427-6437, and U.S. Pat. No.4,889,818 and copending Ser. No. 143,441, filed Jan. 12, 1988, nowabandoned, which are incorporated herein by reference. Crudepreparations of a DNA polymerase activity isolated from T. aquaticushave been described by others (Chien et al., 1976, J. Bacteriol.12.7:1550-1557, and Kaledin et al., 1980, Biokymiya 45:644-651).

The thermostable DNA polymerase from Thermus thermophilus (Tth) has alsobeen purified and is described in commonly assigned, copending Ser. No.455,967, filed Dec. 12, 1989, which is incorporated herein by reference.The '967 patent application also describes that the gene encoding TthDNA polymerase enzyme from Thermus thermophilus has been identified andcloned. Recombinant Tth provides an alternative means for preparing thethermostable enzyme. Crude preparations of DNA polymerase activityisolated from T. thermophilus have been described by Ruttiman et al.,1985, Eur. J. Biochem, 149:41-46. The thermostable DNA polymerase fromThermotoga maritima has been identified and cloned and is described incopending Ser. No. 567,244, filed Aug. 13, 1990, now abandoned, andincorporated herein by reference.

PCR requires a nucleic acid template and appropriate primers foramplification. The DNA to be amplified may be synthetic or genomic,contained in a plasmid, or contained in a heterogenous sample. If thenucleotide sequence to be amplified is RNA, the nucleic acid molecule isfirst treated with reverse transcriptase in the presence of a primer toprovide a cDNA template for amplification. Prior to the presentinvention, amplification of RNA necessitated a reverse transcriptionstep with, e.g., a non-thermostable reverse transcriptase such as MolonyMurine Leukemia Virus Reverse Transcriptase (MoMuLV RT) or AMV-RT,followed by treatment of the resulting single-stranded cDNA with a DNApolymerase. The amplification of RNA could be greatly simplified by theavailability of a method for reverse transcribing RNA and amplifying DNAwith a single enzyme.

Taq polymerase has been reported to inefficiently synthesize cDNA usingMg⁺² as the divalent metal ion (Jones and Foulkes, 1989, Nuc. Acids.Res. 176:8387-8388). Tse and Forget, 1990, Gene 88:293-296; and Shafferet al., 1990, Anal. Biochem. 190:292-296, have described methods foramplifying RNA using Taq polymerase and Mg⁺² ion. However, the methodsare inefficient and insensitive. For example, Tse and Forget demonstratethat 4 μg of total RNA is required to generate sufficient PCR productfor ethidium bromide-stained gel visualization, using an abundantlyexpressed mRNA target.

The present invention addresses this need and provides high temperaturecDNA synthesis by thermoactive DNA polymerases. The present inventionprovides improved methods for a one enzyme, one tube, coupled reversetranscription/amplification assay using a thermostable DNA polymerase.The need to open the reaction vessel and adjust reaction componentsbetween the two steps is eliminated. The methods offer enhancedsensitivity, simplicity, and specificity over current methods.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method for amplifying a targetRNA molecule in a sample, the method comprising the steps of: (a)treating the sample in a reaction mixture comprising a first and secondprimer, wherein the first primer is sufficiently complementary to thetarget RNA to hybridize therewith and initiate synthesis of a cDNAmolecule complementary to the target RNA, and the second primer issufficiently homologous to the target RNA to hybridize to the cDNA andinitiate synthesis of an extension product, and a thermostable DNApolymerase in the presence of all four deoxyribonucleosidetriphosphates, in an appropriate buffer, wherein the buffer comprisesMn⁺², at a temperature sufficient for the thermostable DNA polymerase toinitiate synthesis of an extension product of the first primer toprovide a cDNA molecule complementary to the target RNA; (b) treatingthe reaction mixture at an appropriate temperature to providesingle-stranded cDNA; (c) treating the reaction mixture at anappropriate temperature for the thermostable DNA polymerase to initiatesynthesis of an extension product of the second primer to provide adouble-stranded cDNA molecule; and (d) amplifying the double-strandedcDNA molecule of step (c) by a polymerase chain reaction.

The present invention provides methods for sterilizing reversetranscription reactions, amplification reactions, and homogeneousreverse transcription/amplification reactions, contaminated with nucleicacids generated from previous reverse transcription, amplification,and/or homogeneous reverse transcription/amplification reactions. Forexample the invention provides a method of sterilizing a reversetranscription reaction contaminated with nucleic acids generated from aprevious reverse transcription wherein the previous reversetranscription resulted from mixing conventional and unconventionalnucleoside triphosphates into a reverse transcription reaction mixtureand generating cDNA products having the conventional and unconventionalnucleotides incorporated therein, which method comprises degrading thecontaminating nucleic acids by hydrolyzing covalent bonds of theunconventional nucleotides.

In another aspect, the invention provides a method of sterilizing areverse transcription reaction contaminated with nucleic acids generatedfrom a previous homogeneous reverse transcription/amplification reactionwherein the previous homogeneous reaction resulted from mixingconventional and unconventional nucleoside triphosphates into ahomogeneous reverse transcription/amplification reaction mixture andgenerating cDNA and amplified products having the conventional andunconventional nucleotides incorporated therein, which method comprisesdegrading the contaminating amplified products by hydrolyzing covalentbonds of the unconventional nucleotides.

In one embodiment this method encompasses degrading the contaminatingnucleic acid product with uracil-DNA glycosylase in an aqueous solutioncontaining a target nucleic acid sequence; which further comprisesinactivating the glycosylase in the presence of the target nucleic acidsequences (such as by heat denaturation); and, reverse transcribing andamplifying the target sequence by a thermostable DNA polymerase. Thedegradation of the contaminating product may be accomplished while theproduct is in contact with a nucleic acid reversetranscription/amplification reaction system. Thus, one can prepare asample for reverse transcription/amplification, treat the sample by thepresent method to degrade any contaminating nucleic acid generated by aprevious reverse transcription, amplification, and/or homogeneousreverse transcription/amplification reaction, and then amplify thetarget nucleic acid in the sample without having to adjust reactionvolume or composition between steps.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph comparing reverse transcriptase activity of E. coliPol I, and Taq polymerase in MgCl₂ and MnCl2 buffers. To the right, theactivity attributable to 10 units, 1 units, 0.1 units and 0.01 units ofMoMuLV-RT is shown as a standard.

FIG. 2 is a graph comparing reverse transcription of an RNA-template, atvarying Mn⁺² concentrations, catalyzed by Tth polymerase, 94 kDa rTaqDNA polymerase, and AmpliTaq™ DNA polymerase, Stoffel Fragment (alsoreferred to as 62 kDa rTaq).

FIG. 3 depicts the results of the coupled RT/PCR assay described inExample V.

FIG. 4 depicts the results of the coupled RT/PCR assay described inExample VI using various amounts of total cellular RNA.

FIG. 5 depicts the results of an RT/PCR assay where differentthermostable enzymes are employed for the RT and PCR assays.

DETAILED DESCRIPTION

The present invention provides improved methods for efficientlytranscribing and amplifying RNA. These improvements are achieved by thediscovery and application of previously unknown properties ofthermoactive DNA polymerases. The methods provide a one enzyme procedurefor reverse transcribing and amplifying any desired RNA target andreplace prior methods requiring more than one enzyme. Methods areprovided for a coupled, one tube procedure that eliminates the need toopen the reaction vessel for modifying reaction components between thetranscription and amplification steps. The invention also providesmethods for minimizing the effects of carryover contamination of RNAreverse transcription/amplification assays due to reverse transcribed oramplified products from previous reactions.

The methods comprise treating a sample containing said RNA template withan oligonucleotide primer, which primer is sufficiently complementary tosaid RNA template to hybridize therewith, and a thermoactive DNApolymerase in the presence of all four deoxyribonucleosidetriphosphates, in an appropriate buffer and at a temperature sufficientfor said primer to hybridize to said RNA template and said thermoactiveDNA polymerase to catalyze the polymerization of saiddeoxyribonucleoside triphosphates to form a cDNA sequence complementaryto the sequence of said RNA template. According to the invention, theDNA polymerase may be thermostable as well as thermoactive.

In another aspect, a primer suitable for annealing to an RNA templatemay also be suitable for amplification by PCR. For PCR, a second primer,complementary to the reverse transcribed cDNA strand, provides a sitefor initiation of synthesis of an extension product. As is well known,the thermostable DNA polymerase is able to catalyze this extensionreaction on the DNA template; however, until the present invention, noone recognized that the enzyme could also catalyze the RNA-dependentreverse transcription reaction.

In the amplification of an RNA molecule by a thermoactive DNApolymerase, the first extension reaction is reverse transcription usingan RNA template, and a DNA strand is produced. The second extensionreaction, using the DNA template, produces a double-stranded DNAmolecule. Thus, synthesis of a complementary DNA strand from an RNAtemplate by a thermoactive DNA polymerase provides the starting materialfor amplification.

In another aspect of the invention, a thermostable DNA polymerase can beused in a coupled, one-enzyme reverse transcription/amplificationreaction. Methods are provided for both non-homogeneous and homogeneousRT/PCR assays. The term "homogeneous" as used herein refers to atwo-step single addition reaction for reverse transcription andamplification of an RNA target. By homogeneous it is meant thatfollowing the reverse transcription step, there is no need to open thereaction vessel or otherwise adjust reaction components prior to theamplification step. In a non-homogeneous RT/PCR reaction, followingreverse transcription and prior to amplification any one or more of thereaction components is adjusted, added, or diluted including enzyme,primers, divalent cation, salts, pH, or dNTPs.

The term "homogeneous reverse transcription/amplification reactionmixture" refers to an aqueous solution comprising the various reagentsused to reverse transcribe and amplify a target RNA. These includeenzymes, aqueous buffers, salts, oligonucleotide primers, target nucleicacid, and nucleoside triphosphates. Depending upon the context, themixture can be either a complete or incomplete homogeneous reversetranscription/amplification reaction mixture.

The present invention provides simplified and improved methods fordetecting RNA target molecules in a sample. These methods employthermostable DNA polymerases to catalyze reverse transcription, secondstrand cDNA synthesis, and, if desired, amplification. Thus, theinvention provides methods which require only one enzyme where previousmethods required two. Prior methods also required two sets of incubationconditions, necessitated by the use of different enzymes for eachprocedure. The methods of the present invention provide RNAtranscription and amplification with significantly enhanced specificityand with fewer steps than previous RNA cloning and diagnostic methods.These methods are adaptable for use in kits for laboratory or clinicalanalysis.

The term "reverse transcription reaction mixture" refers to an aqueoussolution comprising the various reagents used to reverse transcribe atarget RNA. These include enzymes, aqueous buffers, salts,oligonucleotide primers, target nucleic acid, and nucleosidetriphosphates. Depending upon the context, the mixture can be either acomplete or incomplete reverse transcription reaction mixture.

For amplification of the cDNA product a number of methods are availableto one of ordinary skill in the art. As used herein the term"amplification reaction system" refers to any in vitro means formultiplying the copies of a target sequence of nucleic acid. Suchmethods include but are not limited to polymerase (PCR), DNA ligase,(LCR), Qβ RNA replicase, and RNA transcription-based (TAS and 3SR)amplification systems.

This invention is not limited to any particular amplification system. Asother systems are developed, those systems may benefit by practice ofthis invention. A recent survey of amplification systems was publishedin Bio/Technology 8:290-293, April 1990, incorporated herein byreference.

The term "amplification reaction mixture" refers to an aqueous solutioncomprising the various reagents used to amplify a target nucleic acid.These include enzymes, aqueous buffers, salts, amplification primers,target nucleic acid, and nucleoside triphosphates. Depending upon thecontext, the mixture can be either a complete or incompleteamplification reaction mixture. In the preferred embodiment of theinvention the amplification system is PCR and the amplification reactionmixture is a PCR mixture.

The present invention is suitable for transcribing and amplifying RNAfrom a number of sources. The RNA template may be contained within anucleic acid preparation from an organism, for example, a viral orbacterial nucleic acid preparation. The preparation may contain celldebris and other components, purified total RNA, or purified mRNA. TheRNA template may be a population of heterogeneous RNA molecules in asample or a specific target RNA molecule.

RNA suitable for use in the present methods may be contained in abiological sample suspected of containing a specific target RNA. Thebiological sample may be a heterogenous sample in which RNA is a smallportion of the sample, as in for example, a blood sample or a biopsiedtissue sample. Thus, the method is useful for clinical detection anddiagnosis. The RNA target may be indicative of a specific disease orinfectious agent.

RNA is prepared by any number of methods; the choice may depend on thesource of the sample and availability. Methods for preparing RNA aredescribed in Davis et al., 1986, Basic Methods in Molecular Biology,Elsevier, N.Y., Chapter 11; Ausubel et al., 1987, Current Protocols inMolecular Biology, Chapter 4, John Wiley and Sons, NY; Kawasaki andWang, 1989, PCR Technology, ed. Erlich, Stockton Press NY; Kawasaki,1990, PCR Protocol: A Guide to Methods and Applications, Innis et al.eds. Academic Press, San Diego; and Wang and Mark, 1990, PCR Protocols:A Guide to Methods and Applications, Innis et al. eds. Academic Press,San Diego; all of which are incorporated herein by reference.

In an illustrative embodiment, the RNA template was synthesized in vitroby T7 RNA polymerase transcription from a DNA template. The resultingRNA molecule, referred to as cRNA, may be purified by various meansincluding gel electrophoresis or oligo(dT) chromatography (see Wang etal., 1989, Proc. Natl. Acad. Sci. 86:9717, and commonly assigned, U.S.Pat. No. 5,219,727, filed Sep. 28, 1989, and issued on Jun. 15, 1993,incorporated herein by reference).

The first step of the present method requires that the RNA template iscombined with a suitable primer. As used herein the term "primer" refersto an oligonucleotide capable of acting as a point of initiation of DNAsynthesis when annealed to a nucleic acid template under conditions inwhich synthesis of a primer extension product is initiated, i.e., in thepresence of four different nucleoside triphosphates and a thermostableenzyme in an appropriate buffer ("buffer" includes pH, ionic strength,cofactors, etc.) and at a suitable temperature. A suitable primer usefulin step (a) of the disclosed methods can hybridize to an RNA template. Aprimer comprising a sequence sufficiently complementary to a specificRNA target molecule may be used to prime synthesis of the first cDNAstrand complementary to a specific target RNA segment if present. Theprimer is sufficiently long to prime the synthesis of extension productsin the presence of the thermostable enzyme. The primer may be anoligodeoxyribonucleotide such as oligo(dT).

Oligo(dT) hybridizes to the polyadenylation (polyA) sequence of mRNAsand provides a primer for cDNA synthesis from a heterogeneous populationof mRNAs. Because most eukaryotic mRNA molecules contain a polyAsequence at the 3' end, an oligo(dT) primer has general utility in thepresent methods, for example, in the preparation of a cDNA library.

The primer typically contains 10-35 nucleotides, although that exactnumber is not critical to the successful application of the method.Short primer molecules generally require lower temperatures to formsufficiently stable hybrid complexes with the template. For oligo(dT) aprimer 16-21 nucleotides in length is suitable for high temperature cDNAsynthesis according to the disclosed methods; however, it may bepreferable to provide an initial incubation at suboptimal temperature toelongate the oligo(dT) primer, thus providing enhanced stability of theprimer-template duplex. For example, although Tth pol is only marginallyactive at temperatures low enough for d(T)₁₆ to anneal, the enzyme hassufficient RT activity to extend d(T)₁₆ on an RNA template at 42° C.Thus, a preferred method for high temperature reverse transcriptionusing oligo(dT)₁₆₋₂₁ includes a 5-10 minute room temperature incubation,generally carried out as part of setting up the reaction, followed by 10minutes at 42° C. and finally 2.5-15 minutes at 70° C. Alternatively,low temperature incubations can be avoided by using oligo(dT) ofincreased chain length (i.e., oligo(dT)₃₅₋₄₅). In the present examplesof the invention the primers are DNA complementary to a portion of themRNA molecules encoding the human cytokines interleukin-1-alpha (IL-1α)or interleukin-1-beta (IL-1β). In several examples, the cDNA primerhybridizes to a synthetic RNA template (cRNA).

Synthetic oligonucleotides can be prepared using the triester method ofMatteucci et al., 1981,I. Am. Chem. Soc. 103:3185-3191. Alternativelyautomated synthesis may be preferred, for example, on a Biosearch 8700DNA Synthesizer using cyanoethyl phosphoramidite chemistry.

For primer extension to occur this primer must anneal to the RNAtemplate. Not every nucleotide of the primer must anneal to the templatefor reverse transcription to occur. The primer sequence need not reflectthe exact sequence of the template. For example, a non-complementarynucleotide fragment may be attached to the 5' end of the primer with theremainder of the primer sequence being complementary to the RNA.Alternatively, non-complementary bases can be interspersed into theprimer, provided that the primer sequence has sufficient complementaritywith the RNA template for hybridization to occur and allow synthesis ofa complementary DNA strand.

Prior methods of cDNA preparation required a pre-annealing step.Destabilization of secondary and tertiary structure of the RNA templatemay be required to allow the primer to hybridize to the RNA. Generally,annealing is accomplished by various means and is routinely accomplishedin the presence of an annealing buffer. Maniatis et at. (supra) provideexamples of annealing buffers. Annealing methods include, but are notlimited to, incubating the RNA/primer mixture at a high temperature fora short period of time followed by step-wise cooling or quick chillingthe mixture in a dry ice/ethanol bath. To prevent intra-strand secondarystructure interactions from interfering with cDNA synthesis or primerannealing, at the low temperatures used previously for reversetranscription, some investigators modify the RNA template by treatmentwith chemical denaturants such as methylmercury hydroxide (Baily andDavidson, 1976, Anal. Biochem. 70:75). However, such denaturants aregenerally highly toxic, carcinogenic compounds and must be carefullyremoved to prevent enzyme inhibition.

According to the present invention, although the primer must anneal tothe template for reverse transcription to occur, a separate annealingstep is not a necessity. Because thermoactive reverse transcriptaseactivity is not irreversibly denatured at the high temperaturespreferred for stringent annealing, there is no need for the quick chillor step-wise cooling of the denatured template, prior to the addition ofthe polymerase. Prior methods necessitated that the heated, denaturedRNA was cooled in a manner that would maintain the annealedprimer-template structure while reducing the temperature to provideconditions compatible with enzyme activity, usually 37°-42° C. Thepresent invention provides methods for high temperature reversetranscription of RNA and eliminates the necessity of a pre-annealingstep and the use of chemical denaturants. This aspect of the inventionis exemplified in Examples V-XI.

The present methods provide that reverse transcription of the annealedprimer-RNA template is catalyzed by a thermoactive or thermostable DNApolymerase. As used herein, the term "thermostable polymerase" refers toan enzyme that is heat stable or heat resistant and catalyzespolymerization of deoxyribonucleotides to form primer extension productsthat are complementary to a nucleic acid strand. Thermostable DNApolymerases useful herein are not irreversibly inactivated whensubjected to elevated temperatures for the time necessary to effectdestabilization of single-stranded nucleic acids or denaturation ofdouble-stranded nucleic acids during PCR amplification. Irreversibledenaturation of the enzyme refers to substantial loss of enzymeactivity. Preferably a thermostable DNA polymerase will not irreversiblydenature at about 90°-100° C. under polymerization conditions.

In another aspect of the invention, it is only essential that the DNApolymerase for high temperature reverse transcription is thermoactive.As used herein, the term "thermoactive polymerase" refers to an enzymethat is capable of efficiently catalyzing polymerization ofdeoxyribonucleotides to form a primer extension product complementary toa nucleic acid template strand at temperatures above 60° C. According tothe present invention, thermoactive polymerases for reversetranscription have maximal activity over 50° C. The thermoactive DNApolymerase will not irreversibly denature at temperatures between 50°C.-80° C. under conditions for RNA destabilization and primer annealing.

In the examples provided, the thermoactive DNA polymerases are alsothermostable; however, a thermoactive, non-thermostable enzyme is alsosuitable for practicing the present invention. Because the preparationof cDNA from an RNA template does not involve repeated denaturationcycles at elevated temperatures, it is not essential that enzymes usefulin the method are thermostable as well as thermoactive. However, in oneembodiment of the invention, a homogeneous RT/PCR procedure is provided.Because the reaction components are not adjusted between the RT and PCRsteps, a thermostable DNA polymerase is preferred.

The heating conditions will depend on the buffer, salt concentration,and nucleic acids being denatured. Of course, it will be recognized thatfor the reverse transcription of mRNA, the template molecule isgenerally single-stranded and, therefore, a high temperaturedenaturation step is unnecessary. However, double-stranded RNA alsoprovides a suitable template for the reverse transcription/amplificationmethods described, following an initial denaturation orstrand-separation step. Double-stranded RNA templates may include, forexample, Reo virus, blue tongue virus, Colorado tick fever virus, andyeast killer factor.

Temperatures for RNA destabilization typically range from 50°-80° C. Afirst cycle of primer elongation provides a double-stranded templatesuitable for denaturation and amplification as referred to above.Temperatures for nucleic acid denaturation typically range from about90° to about 105° C. for a time sufficient for denaturation to occur,which depend on the nucleic acid length, base content, andcomplementarity between single-strand sequences present in the sample,but typically about 0.5 to 4 minutes.

The thermostable or thermoactive DNA polymerase preferably has optimumactivity at a temperature higher than about 40° C., e.g., 60°-80° C. Attemperatures much above 42° C., DNA and RNA-dependent polymerases, otherthan thermostable or thermoactive DNA polymerases, are inactivated.Shimomave and Salvato, 1989, Gene Anal. Techn. 6:25-28, describe that at42° C. AMV-RT has maximum activity. At 50° C. the enzyme has 50%activity, and at 55° C. AMV-RT retains only 10% of its optimal level ofactivity. Thus, AMV-RT is inappropriate for catalyzing high temperaturepolymerization reactions utilizing an RNA template. Only the presentmethod provides methods for efficient high temperature reversetranscription with thermoactive DNA polymerases.

Hybridization of primer to template depends on salt concentration aswell as composition and length of primer. When using a thermostable orthermoactive polymerase, hybridization can occur at higher temperatures(e.g., 45°-70° C.) which are preferred for increased selectively and/orhigher stringency of priming. Higher temperature optima for thepolymerase enzyme enable RNA reverse transcription and subsequentamplification to proceed with greater specificity due to the selectivityof the primer hybridization process. Preferably, the optimum temperaturefor reverse transcription of RNA ranges from about 55°-75° C., morepreferably 65°-70° C.

The present invention provides a method for reverse transcription of anRNA template, having enhanced primer directed specificity, catalyzed bya thermostable DNA polymerase. The methods disclosed are improved overprior methods for the reverse transcription of RNA. These methodsprovide for the amplification of an RNA segment via an RNA/cDNA hybridintermediate molecule. The hybrid molecule is a suitable template foramplification by PCR. Thus, the reverse transcription and amplificationreactions are coupled. Previous RNA amplification methods requireincubation of the RNA/primer mixture in the presence of reversetranscriptase at 37° C.-42° C. prior to the initiation of anamplification reaction. Only by the present invention are all of theenzymatic steps for RNA amplification catalyzed by a thermostable DNApolymerase. The advantages brought to PCR by the commercial availabilityof Taq and Tth polymerases, the disclosed methods for preparing Tthpolymerase, and the commercial availability of Tth DNA polymerasereverse transcription/DNA amplification kits (Perkin Elmer CetusInstruments) are now, by the methods disclosed herein, applicable toreverse transcription, RNA detection, cDNA preparation and coupledreverse transcription/cDNA amplification of RNA.

DNA amplification procedures by PCR are well known and are described inU.S. Pat. No. 4,683,202. For ease of understanding the advantagesprovided by the present invention, a summary of PCR is provided. PCRrequires two primers that hybridize with the double-stranded targetnucleic acid sequence to be amplified. In PCR, this double-strandedtarget sequence is denatured and one primer is annealed to each strandof the denatured target. The primers anneal to the target nucleic acidat sites removed from one another and in orientations such that theextension product of one primer, when separated from its complement, canhybridize to the other primer. Once a given primer hybridizes to thetarget sequence, the primer is extended by the action of a DNApolymerase. The extension product is then denatured from the targetsequence, and the process is repeated.

In successive cycles of this process, the extension products produced inearlier cycles serve as templates for DNA synthesis. Beginning in thesecond cycle, the product of amplification begins to accumulate at alogarithmic rate. The amplification product is a discretedouble-stranded DNA molecule comprising: a first strand which containsthe sequence of the first primer, eventually followed by the sequencecomplementary to the second primer, and a second strand which iscomplementary to the first strand.

Due to the enormous amplification possible with the PCR process, smalllevels of DNA carryover from samples with high DNA levels, positivecontrol templates or from previous amplifications can result in PCRproduct, even in the absence of purposefully added template DNA. Ifpossible, all reaction mixes are set up in an area separate from PCRproduct analysis and sample preparation. The use of dedicated ordisposable vessels, solutions, and pipettes (preferably positivedisplacement pipettes) for RNA/DNA preparation, reaction mixing, andsample analysis will minimize cross contamination. See also Higuchi andKwok, 1989, Nature, 339:237-238 and Kwok, and Orrego, in: Innis et al.eds., 1990 PCR Protocols: A Guide to Methods and Applications, AcademicPress, Inc., San Diego, Calif., which are incorporated herein byreference.

One particular method for minimizing the effects of cross contaminationof nucleic acid amplification is described in U.S. Ser. No. 609,157,filed Nov. 2, 1990, now abandoned, which is incorporated herein byreference. The method involves the introduction of unconventionalnucleotide bases, such as dUTP, into the amplified product and exposingcarryover product to enzymatic and/or physical-chemical treatment torender the product DNA incapable of serving as a template for subsequentamplifications. For example, uracil-DNA glycosylase, also known asuracil-N-glycosylase or UNG, will remove uracil residues from PCRproduct containing that base. The enzyme treatment results indegradation of the contaminating carryover PCR product and serves to"sterilize" the amplification reaction.

The term "unconventional" when referring to a nucleic acid base,nucleoside, or nucleotide includes modifications, derivations, oranalogs of conventional bases, nucleosides or nucleotides whichnaturally occur in a particular polynucleotide (e.g., DNA [dA, dG, dC,dT] or RNA [A, G, C, U]). Uracil is a conventional base in RNA (i.e.,covalent attachment to ribose in a ribopolynucleotide) but anunconventional base in DNA (i.e., covalent attachment to deoxyribose ina deoxyribopolynucleotide). In a coupled RT/PCR reaction, it isdesirable to sterilize the reaction prior to the RT step to eliminatecarryover nucleic acid products of prior reverse transcription and/oramplification reactions. Sterilization after reverse transcription, andprior to PCR, results in degradation of non-contaminating cDNA productscontaining dUTP, as well as contaminating product. Synthesis of cDNA inthe presence of dTTP and absence of dUTP is impractical. For efficientincorporation of dUTP into the subsequent PCR product, a vast excess ofdUTP would be required to dilute the dTTP present as carryover from thereverse transcription step. Furthermore, this would require opening thetube in order to add the dUTP. Consequently, the effectiveness of UNGsterilization would be diminished.

The present invention provides methods for sterilization of the RT/PCRreaction. Example XI demonstrates this aspect of the invention. Whenunconventional nucleosides are being incorporated into amplificationproducts, routine titration experiments are useful to optimize reactionconditions, and U.S. Ser. No. 609,157, filed Nov. 2, 1990, nowabandoned, provides guidance for incorporating unconventionalnucleotides. The parameters which are varied include, but are notlimited to the concentration of divalent cation, pH range, concentrationof polymerase enzyme, concentration of the unconventional nucleoside,the addition of natural nucleoside for which the unconventionalnucleoside is inserted, time of each cycle, and temperature.

Generally, the concentration of dNTPs in a PCR is within the range20-200 μM each dNTP. For incorporating dUTP the efficiency ofamplification is improved at an elevated nucleotide concentration. InExample XI, the concentration of dNTP in the PCR is 200 μM and dCTP,dGTP, and dATP are also present at the same concentration, although thisis not essential. The concentration of MgCl₂ is increased accordingly,on an equimolar basis, when the concentration of dNTP is increased. InExample XI, the PCR contains 200 μM of each dGTP, dATP, dUTP, and dCTPand 2 mM MgCl₂, and provides efficient amplification.

For reverse transcription using a thermostable polymerase, Mn⁺² ispreferred as the divalent cation. Mn⁺² is included as a salt, forexample, MnCl₂. The MnCl₂ is generally present at a concentration of0.5-7.0 mM, and 0.8-1.4 mM is preferred when 200 μM of each dGTP, dATP,dUTP, and, dCTP are utilized; however, 1.2 mM MnCl₂ is most preferred.As noted above for amplification, the optimal concentration of theunconventional nucleotide, and divalent cation may vary in the reversetranscription reaction, depending on the total dNTP concentration and onthe particular primers, template, and polymerase present.

In one embodiment of the invention, at Example X, a two-step singleaddition procedure is provided for coupled RT/PCR. In the method,following reverse transcription, there is no buffer adjustment requiredprior to PCR. Manganese serves as the divalent cation for both the RTand PCR steps. For incorporating either dTTP or dUTP, using 200 mMdNTPs, the concentration of MnCl₂ is lowered to avoid a reduction inamplification efficiency that may occur when MnCl₂ concentration ismaintained at 1.2 mM during PCR.

Following amplification and analysis of the RT/PCR result, the RT/PCRproduct may be introduced unintentionally as a contaminant in otherreactions. Prior to subsequent RT, RT/PCR or amplification reactions,the reaction mixtures are treated with a DNA glycosylase specific forthe unconventional nucleotide incorporated during the prior RT/PCR. Inthis manner, any previous RT/PCR product, present as a contaminant insubsequent RT, RT/PCR or amplification reaction mix containing a targetnucleic acid, is hydrolyzed.

Consequently, in practice, the sterilization treatment is carried outprior to the RT/PCR assay to eliminate carryover of dUTP containingproduct DNAs. For example, prior to the 70° C. incubation of the reversetranscription reaction mix, 0.2-1.0 units UNG per 20 μl RT/reaction isadded and incubated for 1-10 minutes at room temperature. The subsequent70° C. RT and 95° C. denaturation steps serve to inactivate UNG so thatnewly synthesized cDNA and PCR products are not degraded. UNG iscommercially available from Perkin Elmer Cetus Instruments. U.S. Ser.No. 609,157 now abandoned, describes methods for producing UNG byrecombinant means and also thermolabile UNG derivates which do notregain activity after heating above the denaturation temperature of theDNA sample. Such derivates may be preferred for practicing the presentinvention.

The target of amplification can be an RNA/DNA hybrid molecule. Thetarget can be a single-stranded or double-stranded nucleic acid.Although the PCR procedure described above assumed a double-strandedtarget, this is not a necessity. After the first amplification cycle ofa single-stranded DNA target, the reaction mixture contains adouble-stranded DNA molecule consisting of the single-stranded targetand a newly synthesized complementary strand. Similarly, following thefirst amplification cycle of an RNA/cDNA target, the reaction mixturecontains a double-stranded cDNA molecule. At this point, successivecycles of amplification proceed as described above. In the presentmethods, the target of amplification is a single-stranded RNA, and thefirst amplification cycle is the reverse transcription step.Alternatively, if the starting template is double-stranded RNA, aninitial high temperature denaturing step may be used to preparesingle-stranded RNA template.

As used herein the term "cDNA" refers to a complementary DNA moleculesynthesized using a ribonucleic acid strand (RNA) as a template. The RNAmay be mRNA, tRNA, rRNA, or another form of RNA, such as viral RNA. ThecDNA may be single-stranded, double-stranded or may be hydrogen-bondedto a complementary RNA molecule as in an RNA/cDNA hybrid.

The methods of the present invention provide means for obtaining cDNAfrom a desired RNA template wherein the desired end product is producedwith greater specificity than by previous methods. Additionally, thepresent invention provides that cDNA synthesis can be coupled toamplification by PCR. These methods incorporate previously unknownproperties of thermoactive DNA polymerases. In the disclosedembodiments, methods are provided which utilize Taq and Tth polymerasesfor reverse transcription. These embodiments should not be construed asa limitation of the present invention.

Thermostable polymerases are available from a number of sources. Theenzyme may be a native or recombinant protein. A preferred thermostableenzyme is Thermus thermophilus DNA polymerase (Tth polymerase), purifiedfrom Thermus thermophilus (see copending Ser. No. 455,967, now abandonedin favor of continuation application U.S. Ser. No. 07/880,478, filed May6, 1992, and published as PCT Patent Publication No. WO 91/09950, whichis incorporated herein by reference). Alternatively, Tth is purifiedfrom recombinant host cells as described herein and may be designated asrTth. Also preferred for practicing the invention is Taq polymerase. Taqis commercially available as a recombinant product or purified as nativeTaq from Thermus aquaticus (Perkin Elmer-Cetus Inst.). As used herein,recombinant Taq may be designated as rTaq and native Taq may bedesignated as nTaq.

An important aspect of the present invention relates to Tth DNApolymerase for reverse transcription and amplification of nucleic acids.Tth polymerase is commercially available from Perkin Elmer CetusInstruments. The gene encoding this enzyme has been cloned from T.thermophilus genomic DNA. Tth polymerase has a predicted molecularweight of about 94 kDa, based on the inferred amino acid sequence. Thecomplete coding sequence (˜2.5 kb) for the Tth polymerase can be easilyobtained in an ˜3.7 kilobase (kb) HindlII-BstEII restriction fragment ofplasmid pBSM: Tth, although this ˜3.7 kb fragment contains an internalHindIII restriction enzyme site. One specific isolate of pBSM:Tth in E.coli K12 strain DG101 was purified and referred to as pBSM:Tth 10. Thisplasmid was deposited with the American Type Culture Collection (ATCC)in host cell E. coli K12 strain DG101 on Dec. 21, 1989, under ATCCaccession No. 68195. The availability of the Tth DNA polymerase genesequence provides the necessary starting material for one skilled in theart to prepare any number of expression vectors applicable to a varietyof host systems for preparing recombinant Tth DNA polymerase. Similarly,mutant forms of the polymerase may be prepared that retain the DNApolymerase activity and are within the meaning of the term Thermusthermophilus DNA polymerase.

A number of Tth DNA polymerase expression vectors are described incopending Ser. No. 455,967, now abandoned, which are suitable forproducing recombinant purified Tth for use in the present invention, andthat application is incorporated herein by reference. Of theseexpression vectors, plasmid pLSG33 E. coli K12 strain DG116 was used asa source of recombinant Tth. In plasmid pLSG33, expression of the geneencoding Tth polymerase is regulated by the γP_(L) promoter.Construction of pLSG33 is described in detail in co-pending Ser. No.455,967, now abandoned in favor of continuation application U.S. Sr. No.07/880,478, filed May 6, 1992, and also published as PCT PatentPublication No. WO 91/09950, and incorporated herein by reference. Inthat description pBSM:Tth is utilized as the source of the Tth gene.

Once the Tth DNA polymerase has been expressed in a recombinant hostcell, the enzyme can be purified and used in the methods disclosedherein. Purification procedures have been previously described fornative Tth and native and recombinant Taq in Ser. No. 455,611, filedDec. 22, 1989, which issued as U.S. Pat. No. 5,322,770, and Ser. No.143,441, filed Jan. 12, 1988, now abandoned, and the disclosures ofwhich are incorporated herein by reference. Purification of recombinantTth polymerase is generally similar, and the previously describedprocesses are suitable. However, a preferred method for purifyingrecombinant Tth is provided in Example I in the present specification.The procedure for purifying recombinant Tth is simplified over thenative Tth purification scheme. Because the non-native host cell doesnot produce Tth endonuclease I, which co-elutes with native Tthpolymerase, the steps taken to remove TthHB8I endonuclease are notneeded.

Although the present invention is exemplified by Taq and Tth DNApolymerases, the invention is not limited to that description. Otherthermostable polymerases that have been reported in the literature willalso find use in the practice of the methods described. Examples ofthese include polymerases extracted from the thermophilic bacteriaBacillus stearothermophilus, Thermosipho africanus, Thermotoga maritima,Thermus species SPS17, T. flavus, T. lacteus, T. rubens, T. ruber, andT. species Z05. In addition, thermostable polymerases isolated from thethermophilic archaebacteria include, for example, Desulfurococcusmobilis, Methanobacterium thermoautotrophicum, Methanothennus fervidus,Pyrococcus furious, Pyrodictium occultum, Sulfolobus acidocaldarius, S.solfataricus, Thermococcus litoralis, and Thermoplasma acidophilum

Modified thermostable polymerases may result from proteolyticdegradation or may be produced from a truncated gene. These proteins arealso useful in the practice of the present invention so long as theyfunction to polymerize deoxyribonucleoside triphosphates using an RNAtemplate.

Taq polymerase can be prepared as both a 94 kDa and 61 kDa enzyme. The61 kDa enzyme has been previously referred to as the 62 kDa enzyme (seefor example U.S. Pat. No. 4,889,818) and may be referred to as theStoffel Fragment; however, the Taq 61 kDa, 62 kDa, and Stoffel Fragmentenzyme all refer to the same identity. The Stoffel Fragment is aprocessed form of the 94 kDa enzyme, resulting from proteolytic cleavageof the N-terminal region. Alternatively, the Stoffel fragment enzyme canbe made directly as a recombinant protein. The Stoffel Fragment iscomposed of approximately two-thirds of the carboxy-terminal portion ofthe full length protein. Either form of the enzyme will function toreverse transcribe RNA as described herein. In addition to theN-terminal deletion, individual amino acid residues may be modified byoxidation, reduction, or other derivatization, or the protein may becleaved to obtain fragments that retain activity.

Thus, modification to the primary structure itself by deletion,addition, or alteration of the amino acids incorporated into thesequence during translation can be made without destroying the hightemperature DNA polymerase activity of the protein. Such substitutionsor other alterations result in proteins useful in the methods of thepresent invention. The availability of DNA encoding these sequencesprovides the opportunity to modify the codon sequence to generate mutantprotein forms also having reverse transcriptase activity.

As demonstrated herein, Tth DNA polymerase has high reversetranscriptase activity. However, Tth polymerase, as well as Taqpolymerase, lacks a 3' to 5' exonucleolytic proofreading activity. This3' to 5' exonuclease activity is generally considered to be desirablebecause it allows removal of misincorporated or unmatched bases in thenewly synthesized nucleic acid sequences. Because the lack of aproofreading activity may effect enzyme fidelity, the presence of aproofreading exonuclease would be a novel and potentially usefulproperty for a reverse transcriptase.

Thermotoga maritima DNA polymerase (Tma pol) has 3' to 5' exonucleaseactivity. U.S. patent application Ser. No. 567,244, filed Aug. 13, 1990,now U.S. Pat. No. 5,374,553, and incorporated herein by reference,provides means for isolating and producing Tma polymerase. That patentapplication provides the amino acid and nucleic acid sequences for TmaDNA polymerase and describes the amino acid domains for various enzymeactivities, including the 3' to 5' exonuclease activity, as well as the5' to 3' exonuclease activity.

Accordingly, domain shuffling or construction of chimeric DNApolymerases may be used to provide thermostable DNA polymerasescontaining novel properties. For example, a thermostable chimeric DNApolymerase which has the Y to 5' exonuclease domain of Tma polymeraseincorporated into Tth polymerase can be constructed using "overlap" PCR(Higuchi, 1989, PCR Technology supra.). In this method, the intendedjunction sequence is designed into the PCR primers (at their 5' ends).Following the initial amplification of each individual domain, thevarious products are diluted (ca. 100- to 1,000-fold) and combined,denatured, annealed, extended, and then the final forward and reverseprimers are added for an otherwise standard PCR.

Specifically, the polymerase domain of Tth polymerase (amino acids415-834) is joined to the 5' to 3' and 3' to 5' exonuclease domains ofTma polymerase (amino acids 1-475). For example, a Tth polymeraseexpression vector and a portion of the gene encoding Tma polymerase canbe combined as follows. The expression vector pLSG33 is described incopending Ser. No. 455,967 now abandoned in favor of continuationapplication U.S. Ser. No. 07/880,478, filed May 6, 1992, and publishedas PCT Patent Publication No. WO 91/09950, and contains the geneencoding Tth. Plasmid pTMA5'Nde#3 (subsequently referred to as pTma06),described in co-pending Ser. No. 567,244, filed Aug. 13, 1990, nowabandoned, which is incorporated herein by reference, contains the 5'portion of the gene encoding Tma polymerase. To prepare the plasmid foroverlap PCR, the pLSG33 and pTma06 are linearized with NdeI and used intwo separate PCR amplifications using primers A and B for the Tmapolymerase, and primers C and D for the Tth polymerase. The primers'sequences are:

    __________________________________________________________________________    A SEQ ID NO: 1                                                                          5'-GGCATATGGCTAGACTATTTCTTTTTG-3'                                   B SEQ ID NO: 2                                                                          5'-AGGTTCCGATGAAGTCTGTAGGTGATGTCTG-3'                               C SEQ ID NO: 3                                                                          5'-CTACAGACTTCATCGGAACCTCCTTAAGCG-3'                                D SEQ ID NO: 4                                                                          5'-CCAACCCGCCTCGGCCACGAAGG-3'                                       __________________________________________________________________________

In addition to the region of complementation designed into primers B andC, primer A has an Nde I site incorporated into its 5'-termini. Primer Dcorresponds to a portion of the polymerase domain of Tth and is directlydistal to a BamHI site within the 3' region of the Tth gene. The firstround of PCR generates product AB (1441 bp) and product CD (586 bp).Following the initial amplification of the individual domains, thereactions are diluted approximately 100- to 1000-fold and combined,denatured, annealed, and extended using the final forward and reverseprimers (primers A and D, respectively). The final product, AD, isdigested with Nde I and BamHI to provide a 2006 bp product. This productis then ligated back into the expression vector (following digestion ofthe vector, pLSG33, with Nde I and BamHI) and transformed into anappropriate host. The chimeric protein will contain 895 amino acidresidues.

Tth, Taq, and Tma DNA polymerases also contain a 5' to 3' exonucleaseactivity which may be responsible for RNase H activity. The eliminationor reduction of 5' to 3' exonuclease activity by, for example, sitespecific mutagenesis, may provide a preferred form of thermostable DNApolymerase for cDNA synthesis. A substitution of glutamic acid for aglycine residue at amino acid number 103 of the pol A gene of E. colihas been demonstrated to produce a polypeptide defective in 5' to 3'exonuclease activity (Kingsbury and Helinski, 1973, J. Bacteriol.114:1116-1124; Olivera and Bonhoeffer, 1974, Nature 250:513-514; andJoyce et al., 1985, J. Mol. Biol. 186:283-293). The homologous aminoacid is conserved in Tth polymerase (amino acid number 108). The normalGGG codon is mutated to a GAA codon by PCR to provide a novelthermostable DNA polymerases with improved characteristics for reversetranscription of RNA. Alternatively, changing Tth amino acid number 46from glycine to aspartic acid may also effect the 5'→3' exonucleaseactivity providing a novel enzyme.

The fidelity of viral reverse transcriptases, such as AMV-RT andMoMuLV-RT, is compared to thermoactive reverse transcriptases by astraightforward assay procedure. Plasmid BS+ (Stratagene) is used forsuch an assay. The plasmid encodes an α-complementing β-galactosidaseactivity and can be linearized with Nde I. T3 RNA polymerase is used toprepare a cRNA transcript of the α donor region. After treatment of thecRNA with RNase-free DNase and isolation of the cRNA, the cRNA is usedas a template for a reverse transcription/amplification reaction. Areverse transcription primer complementary to the 3' end of the cDNAcontaining an Nde I sequence at its 5' terminus, and an upstream PCRprimer comprising a Pst I sequence at the 5' termini provide a 752 bpPCR product. The PCR product and the pBS+vector are then digested withNde I and Psi I followed by ligation of the PCR product into the vectorand transformation into a suitable host. The presence of white coloniesindicates that a mutation had occurred during the RT or PCRamplification. The assay provides means for assigning a relative valueto the fidelity of the reverse transcriptase activity of variousenzymes. Specific mutations can be determined by sequence analysis.

The method of high temperature reverse transcription provides novelmeans for the detection of specific RNAs in a sample. This method isuseful in a clinical setting to monitor, for example, retrovirusinfection in children born to AIDS victims or to monitor the expressionof diagnostic proteins. Detection of the reverse transcribed oramplified products can be accomplished by any of a number of knownmeans. Such means include, but are not limited to, hybridization withisotopic or non-isotopically labeled probes in, for example, a dot blotor electrophoretic format. A detection format may include a capturestep, such as a solid support substrate and avidin-biotin label system.U.S. Pat. No. 5,210,015, filed Aug. 6, 1990, and issued on May 11, 1993,incorporated herein by reference, describes a method for use of the 5'to 3' nuclease activity of a nucleic acid polymerase. According to themethod, a labeled nucleic acid probe in a hybridized duplex composed ofa labeled oligonucleotide and a target oligonucleotide is degraded.Labeled fragments are subsequently detected. Detection may also includequantitative analysis to monitor progress of, for example, an infectionor response to a treatment regimen. Alternatively, detection may be forthe purpose of cell typing.

Primers can be designed with convenient restriction enzyme recognitionsequences located at or near the 5' end of the primer. In the formationof the cDNA transcript, so long as the 3' end of the primer ishydrogen-bonded to the target sequence, the resulting double-strandedcDNA molecule would contain a specific restriction enzyme recognitionsequence. Following amplification, using the cDNA as a template, therestriction site could be used to facilitate other procedures, forexample, cloning.

Following reverse transcription of RNA by a thermoactive or thermostableDNA polymerase, the RNA can be removed from the RNA/cDNA hybrid by heatdenaturation or by a number of other known means such as alkali, heat,or enzyme treatment. Enzyme treatment may consist of, for example,treating the RNA/cDNA hybrid with RNase H. RNase H is specific for RNAstrands within an RNA/DNA double-stranded molecule. Tth and Taqassociated RNase H and 5'→3' nuclease activities can facilitatehydrolysis of the RNA template and synthesis of the second DNA strand,as well as primer extension for amplification of the cDNA sequence.Alternatively, exogenous RNase H is added from a commercially availablesource.

The RNase H activity of thermostable polymerases provides means fordistinguishing between RNA and DNA templates in a sample. This isparticularly useful for detecting RNA in the presence of homologousduplex DNA. Where the DNA is free of introns in, for example, proviralHIV DNA in sera or plasma, amplified RNA and DNA may not bedistinguishable by size. However, following reverse transcription,thermostable RNase H activity eliminates the necessity for denaturatingthe RNA/cDNA duplex. Consequently, in the presence of genomic orproviral DNA, only the RNA template is amplified in the first PCR cycle.

In a preferred method for distinguishing between homologous RNA and DNAtemplates, amplification primers are used that will effectively lowerthe denaturation temperature of the PCR product. For example, fordetecting HIV RNA, primer pairs SK462 SEQ ID NO: 5(5'AGTYGGAGGACATCAAGCAGCCATGCAAAT)/SK431 SEQ ID NO: 6 (5'TGCTATGTCAGTTCCCCITGGTrCTCT) and SK38 SEQ ID NO: 7 (5'ATAATCCACCTATCCCAGTAGGAGAAAT)/SK39 SEQ ID NO: 8 (5'TITGGTCCITGTCITATGTCCAGAATGC) generate a PCR product that is denaturedwell below 94° C. Typical denaturation temperatures for PCR are 94°-96°C. At the lowered temperature the double-stranded DNA (i.e. the expected"contaminant") is not denatured and would not be amplified. Methods foraffecting the denaturation temperature of PCR products are described indetail in copending U.S. Ser. No. 718,576, filed Jun. 20, 1991, nowabandoned in favor of U.S. Ser. No. 08/033,072, filed Mar. 10, 1993,which issued as U.S. Pat. No. 5,314,809, and incorporated herein byreference.

Alternatively, unconventional nucleotides are useful for effecting thedenaturation temperature of the PCR product. For example, hydroxymethyldUTP (HmdUTP) naturally occurs in SP01 phage DNA as 5'hydroxymethyluracil (HmUra) in place of thymine (Kallen et al., 1962, J.Mol. Biol. 5:248-250, and Levy and Teebor, 1991, Nuc. Acids Res.19(12):3337). The HmUra containing genome melts at 10° C. less than DNAof corresponding thymine content. Incorporation of HmdUTP into cDNAeffectively lowers the denaturation temperature of the reversetranscribed product, in comparison to the denaturation temperature ofthe homologous DNA. Other modified nucleoside triphosphates capable ofeffecting the Tm of the DNA product (e.g., C7dGTP, 7, 7 deaza-2'deoxyguanosine triphosphate) are also suitable for distinguishing betweenhomologous RNA and DNA templates.

Following removal or melting of the RNA template strand, the remainingcDNA strand can then serve as a template for polymerization of aself-complementary strand, providing a double-stranded cDNA moleculesuitable for additional amplification, detection or other manipulation.The second strand synthesis also requires a primer. A sequence specificprimer can be introduced into the reaction mix to prime second strandsynthesis. Alternatively, a duplex adapter-linker may be ligated to thecDNA or the cDNA may be tailed with a terminal transferase-typeactivity. The second strand primer needs only to hybridize to the tailrather than the specific cDNA sequence (see for example, Frohman inInnis et al. supra.). In the practice of the disclosed methods, it maybe desirable to use a first set of primers to synthesize a specific cDNAmolecule and a second nested set of primers to amplify a desired cDNAsegment. All of these reactions may be catalyzed by the samethermostable DNA polymerase.

DNA polymerases require a divalent cation for catalytic activity. Taborand Richardson, 1989, Proc. Natl. Acad. Sci. USA 86:4076-4080, havereported that Mn²⁺ can be substituted for Mg²⁺ in DNA sequencingmethods. These methods require a DNA template and T7 DNA polymerase orE. coli DNA polymerase I.

Either Mn⁺², Mg⁺², or Co⁺² can activate Taq, Tth, and Tma DNApolymerases; however, Mn⁺² is preferred in the present method. In thedisclosed embodiments of the invention, buffers are provided whichcontain Mn⁺² for nucleic acid reverse transcription from an RNAtemplate. These buffers are improved over previous reverse transcriptionbuffers and result in increased cDNA yields. In particular, practice ofthe present methods using Tth and MnCl₂ for amplifying RNA imparts anincrease in sensitivity of at least 10⁶ fold compared to MgCl₂ andstandard PCR conditions.

For reverse transcription, according to the present invention, theprimer-template mixture is incubated with a thermoactive or thermostablepolymerase under suitable polymerization conditions. These conditionsare provided by a buffer containing a divalent cation, a monovalentcation, all four deoxyribonucleotide triphosphates (dNTPs), and abuffering agent. In the present embodiments, for reverse transcriptionthe divalent cation supplied is MgCl₂, MgOAc, or MnCl₂ in aconcentration ranging from 0.5 mM to 7 mM for MnCl₂ or 0.5 to 10 mMMgCl₂. Preferably, the divalent cation is MnCl₂ at a concentrationbetween 0.5 and 2 mM.

The monovalent cation may be supplied by KOAc, NaCl, or KCl. For KCl theconcentration is between 1-200 mM, preferably the concentration isbetween 40 and 100 mM, although the optimum concentration may varydepending on the polymerase used in the reaction. Optimal reversetranscriptase activity is observed between 50 and 75 mM KCl when Tthpolymerase is used. However, enhanced RT/PCR is observed when the KClconcentration is increased up to 100 mM. For AmpliTaq® DNA polymerase,50 mM KCl is preferred. Deoxyribonucleotide triphosphates are added assolution of the salts of dATP, dCTP, dGTP, dUTP, and dTTP, such asdisodium or lithium salts. In the present methods a final concentrationin the range of 1 μM to 2 mM each is suitable, and 100-600 μM ispreferred, although the optimal concentration of the nucleotides mayvary in the reverse transcription reaction, depending on the total dNTPconcentration and on the particular primers and template. For longerproducts, i.e., greater than 1500 bp, 500 μM each dNTP and 2 mM MnCl₂may be preferred.

In one embodiment of the invention, a method for homogeneous RT/PCR isprovided. This two-step, single addition procedure eliminates the needto open the tube after the addition of initial reagents. Thus, theopportunity for contamination between the RT and PCR steps is removed.Due to the high enzyme concentration required for optimum RT activity, ashort extension cycle is preferred, i.e., 10-30 seconds, during each PCRthermocycle. Because there is no buffer adjustment between the RT andPCR steps, in a homogeneous RT/PCR assay MnCl₂ is preferably less than1.0 mM and, most preferably, 0.8 mM. However, the addition of a metalbuffer such as isocitrate allows higher MnCl₂ to be used. Although inthe presence of dUTP, the RT step is more efficient at the high MnCl₂concentration, the PCR step is less efficient. Most preferably 0.8 mMMnCl₂ is used and the product is detected by means more sensitive thatethidium stained gels; i.e., probe hybridization or incorporation oflabeled, or other detectable nucleotides.

A suitable buffering agent is Tris-HCl, pH 8.3, although the pH may bein the range 8.0-8.8. The Tris-HCl concentration is from 5-50 mM,although 10-20 mM is most preferred. Additionally, EDTA less than 0.5 mMmay be present in the reverse transcription reaction mix. Detergentssuch as Tween-20™ and Nonidet™ P-40 are present in the enzyme dilutionbuffers. A final concentration of non-ionic detergent approximately 0.1%or less is appropriate, however, 0.05%-0.01% is preferred and will notinterfere with polymerase activity. Similarly, glycerol is often presentin enzyme preparations and is generally diluted to a concentration of1-10% in the reaction mix. A mineral oil overlay may be added to preventevaporation.

The present methods require only that RNA is present in the sample. Inan example, a synthetic RNA prepared using a plasmid containing a T7promoter is reverse transcribed and amplified by the methods of thepresent invention. In another example, a heterogeneous population oftotal cellular RNA is used to reverse transcribe and amplify a specificmRNA. For practicing the invention the amount of RNA present in thesample is generally within the range of 0.1 pg to 1 μg. The amountrequired and the results will vary depending on the complexity of thesample RNA and the type of detection utilized. Because of the speciftyof the high temperature reverse transcription reaction, 10 to 10⁸molecules of the target RNA are sufficient to provide up to or exceedingmicrogram quantities of PCR product. In several of the disclosedexamples, amplification products are visualized by ethidium bromidestaining after gel electrophoresis of 5% of the total reaction mix.Thus, the amount of target required would be substantially reduced whenalternative means for assay of the product is utilized. For example,isotopically labeled DNA probes suitable for detecting theelectrophoresed PCR products would increase the sensitivity of detectionand therefore decrease the amount of starting material required (e.g.,1-10⁸ molecules of target RNA in the sample).

Preferably, the amount of RNA present in the reverse transcriptionreaction is 10 pg to 500 ng and most preferably 0.1 to 300 ng. In thispreferred range, 1 to 10 units of thermoactive DNA polymerase issufficient for providing a full length cDNA product. To achievepredominantly full length cDNA, the enzyme to template ratio ispreferably greater than 0.5. When the sample contains more than 300 ngof RNA, it may be desirable to include additional enzyme to ensuretranscription of a full length cDNA product from the RNA template.However, if the reverse transcription reaction is coupled to PCR, theeffect of high enzyme concentration in the PCR reaction should beconsidered. For example when Taq is used as the thermoactive polymerase,high enzyme concentrations can result in non-specific PCR products andreduced yields (see Saiki in PCR Technology Ed. Erlich, 1989, StocktonPress). The potential problems resulting from a high enzymeconcentration, however, are easily avoided by inactivating thethermoactive DNA polymerase between the reverse transcription reactionand the amplification reaction. Inactivation is achieved by incubatingthe cDNA synthesis reaction mix at 99° C. for 3 to 10 minutes. Anappropriate amount of thermostable DNA polymerase is then added back tothe reaction mix, and PCR is conducted as usual. This method is alsosuitable when different thermostable DNA polymerases are used for eachof the two reactions, as exemplified in Example VII.

Once the sample containing RNA has been prepared and the suitable primerand salts have been added, the reaction is incubated with thethermoactive DNA polymerase for 1-60 minutes. Usually, however, areaction time of 2 to 30 minutes is suitable. For a target molecule thatis relatively short (˜300 nucleotides), the reverse transcriptionreaction is preferably incubated for approximately 2-5 minutes. If thetarget molecule is long, or if the ratio of total RNA to target RNA ishigh, e.g., 100 copies of target RNA in the presence of 250 ng of totalRNA, an incubation time of 10-30 minutes is preferred.

It is preferred, but not essential that the thermoactive DNA polymeraseis added to the reverse transcription reaction mix after both the primerand the RNA template are added. Alternatively, for example, the enzymeand primer are added last, or the MnCl₂, or template plus MnCl₂ areadded last. It is generally desirable that at least one component, thatis essential for polymerization not be present, until such time as theprimer and template are both present and the enzyme can bind to andextend the desired primer/template substrate (see U.S. patentapplication Ser. No. 481,501, filed Feb. 16, 1990, now abandoned infavor of continuation application U.S. Ser. No. 07/890,300, filed May27, 1992, which is incorporated herein by reference).

In practicing the present methods the reaction mix is incubated above40° C., although a preferred temperature is 55°-75° C. At thistemperature, the specificity of the primer-template annealing isenhanced over the annealing specificity at a lower temperature, and thethermoactive enzyme has higher activity at the elevated temperature. Theelevated temperature reduces non-specific priming by degraded nativenucleic acid and by incorrect primer-template hybridization.

Following reverse transcription, the RNA template may be degraded oralternatively denatured, providing a template for continuous replicationresulting in an excess of single-stranded DNA molecules. This excess ofsingle-stranded DNA can be detected by standard probe hybridizationtechniques. Thus, the invention provides means for direct detection oftarget segments. The resulting nucleic acid products can be detected bya number of electrophoretic or chromatographic means. The use of aradiolabeled triphosphate is helpful in monitoring the extent of thereaction and the size of products formed, although this is not anessential component of the invention.

The reverse transcription reaction products are suitable as templatesfor amplification by PCR. In one embodiment of the invention, followingthe high temperature reverse transcription incubation, the reversetranscription reaction is adjusted to PCR buffering conditions, and theamplification reaction is initiated following the addition of a secondprimer. PCR buffer is added to maintain the appropriate bufferingcapacity, pH, monovalent cation concentration, and to dilute theconcentration of enzyme and dNTPs to within 20-200 μM each dNTP. MgCl₂is added to a final concentration of 1-3 mM. Preferably, theconcentrations of dNTPs in both the reverse transcriptase and PCRreaction mixes are balanced. Because Mn⁺² can induce hydrolysis of RNAand possibly DNA, as well as diminish PCR amplification when present athigh concentrations, in a preferred embodiment of the invention the Mn⁺²is chelated prior to the PCR amplification. The presence of high amountsof Mn⁺² also may decrease fidelity during amplification, howeverchelating the Mn⁺² avoids this problem. Accordingly, it is preferredthat following the reverse transcription reaction, EGTA is added at aconcentration between about 1-10 times the molar concentration of Mn⁺²to chelate the Mn⁺². In the presence of both Mg⁺² and Mn⁺², EGTApreferentially binds Mn⁺². Low dNTP and Mg⁺² concentrations, asdescribed herein, may also increase fidelity of Tth duringamplification. Glycerol and non-ionic detergent (for example, Tween-20™)may be added to a final concentration of between 1-10% and 0.05%-0.01%,respectively, to increase enzyme stability.

In an alternative embodiment, Mn⁺² is not chelated prior to PCR. PCR canutilize Mn⁺² in place of Mg⁺², although Mg⁺² is preferred as describedabove. In particular, for applications such as, for example, large scalediagnostic screening, the risk of infidelity during amplification andlow level hydrolysis of template may be tolerable in view of thetremendous advantages a homogeneous RT/PCR method provides. The two-stepsingle addition procedure minimizes sample handling and reduces thepotential for cross contamination. Because MnCl₂ effects PCR efficiency,the optimum concentration is preferably titrated by standard means forthe particular reaction components utilized: primers, targetconcentration, dNTP concentration, etc. In present embodiments of ahomogeneous RT/PCR assay, the optimum MnCl₂ concentration isapproximately 0.8 mM.

The methods provided herein have numerous applications, particularly inthe field of molecular biology and medical diagnostics. The reversetranscriptase activity described provides a cDNA transcript from an RNAtemplate. The methods for production and amplification of DNA segmentsfrom an RNA molecule are suitable where the RNA molecule is a member ofa population of total RNA or is present in a small amount in abiological sample. Detection of a specific RNA molecule present in asample is greatly facilitated by a thermoactive or thermostable DNApolymerase used in the methods described herein. A specific RNA moleculeor a total population of RNA molecules can be amplified, quantitated,isolated, and, if desired, cloned and sequenced using a thermoactive orthermostable enzyme as described herein.

The methods and compositions of the present invention are a vastimprovement over prior methods of reverse transcribing RNA into a DNAproduct. When starting with an RNA template, these methods have enhancedspecificity and provide templates for PCR amplification that areproduced more efficiently than by previously available methods. Theinvention provides more specific and, therefore, more accurate means fordetection and characterization of specific ribonucleic acid sequences,such as those associated with infectious diseases, genetic disorders, orcellular disorders.

Those skilled in the art will recognize that the compositions of theinstant invention can be incorporated into kits. Thus, the inventionrelates to kits that contain a thermoactive DNA polymerase as well asinstructions describing the method for using the same for reversetranscribing RNA. In one embodiment such a kit may relate to thedetection of at least one specific target RNA sequence in a sample. Sucha kit would comprise, in addition to the elements listed above, a primercomprising a sequence sufficiently complimentary to a specific targetRNA sequence to hybridize therewith. Diagnostic kits for theamplification and detection of at least one specific RNA sequence in asample may comprise a primer having a sequence sufficiently identicalwith the RNA target to hybridize with the first strand of cDNAsynthesized to prime synthesis of a second cDNA strand. Kits maycontain, in addition to the components listed, the fourdeoxyribonucleotide triphosphates, suitable buffers as described herein,oligo(dT), RNase H, linkers for cloning, as well as one or morerestriction enzymes.

The following examples are offered by way of illustration only andshould not be construed as intending to limit the invention in anymanner.

EXAMPLE 1 Materials and Methods

I. Substrates

A. RNA

RNA was synthesized in vitro using T7 RNA polymerase and a synthetictemplate, pAW106. The template, pAW106, contains a T7 promoter adjacentto a synthetically prepared DNA segment followed by a polyadenylationsequence. The RNA produced, referred to herein as cRNA, was purified byoligo(dT) chromatography. The purified material, 1060 bases in length,contained a portion of interleukin 1 β (IL-1β) mRNA sequence. The RNAconcentration was 0.1 μg/μl which was equivalent to ˜0.286 pmoles/μl.

Alternatively, pAW109 (ATCC No. 68152) was used as a template to preparecRNA, 963 bases in length. Whether pAW106 or pAW109 cRNA was used, thecRNA was prepared and quantitated according to Wang et al. supra. Insome examples pAW109 cRNA was diluted to limit the number of templatemolecules and E. coli ribosomal RNA (Boehringer Mannheim) was added fora total of 60 ng of RNA/reaction.

K562, a Philadelphia-chromosome positive cell line (Lozzio and Lozzio,1975, Blood 45:321-334, and Kawasaki et al., 1988, Proc. Natl. Acad.Sci. USA 85:5698-5702), was used as a source of total cellular RNA. TheRNA was purified according to Kawasaki et al., 1985, Science 235:85-88,and Schwartz et al., 1981, J. Immunol. 126:2104-2108.

B. DNA

A DNA template was provided as a control for monitoring the activity ofDNA polymerase. A solution of activated salmon sperm DNA was prepared ata concentration of 2.5 μl/μl in 10 mM Tris-HCl, pH 8.3, 5 mM KCl, and 50μM EDTA. One reaction contained 6.25 μg of salmon sperm DNA template(2.5 μl). In some examples pAW109 was diluted to limit the number oftemplate molecules and E. coli ribosomal RNA (Boehringher Mannheim) wasadded for a total of 60 ng of RNA/reaction.

II. Oligonucleotide Primers

DM156 was used to prime cDNA synthesis using the pAW106 cRNA template.The primer sequence corresponds to a portion of the human IL-1 β geneand is complementary to human IL-1 β mRNA. The primer concentration was100 pmol/μl.

DM152 and DM151 correspond to a portion of the human IL-1α gene andamplify a 420 base pair segment when IL-1α mRNA (for example, from K562cells) is used as the template. A 308 base pair segment is produced frompAW109 cRNA. DM152 hybridizes to pAW109 cRNA or IL-1α mRNA to prime cDNAsynthesis. DM151 hybridizes to the single-stranded cDNA as the"upstream" amplification primer.

TM01 was used as the "downstream" primer to synthesize a cDNA moleculefrom the pAW109 cRNA template and can hybridize to the 3' untranslatedregion of human IL-1 α mRNA. DM151 and TM01 amplify a 736 base pairsegment of pAW109.

    __________________________________________________________________________    DM156                                                                             SEQ ID NO. 9                                                                           5'-TGGAGAACACCACTTGTTGCTCCA                                      DM151                                                                             SEQ ID NO. 10                                                                          5'-GTCTCTGAATCAGAAATCCTTCTATC                                    DM152                                                                             SEQ ID NO. 11                                                                          5'-CATGTCAAATTTCACTGCTTCATCC                                     TM01                                                                              SEQ ID NO. 12                                                                          5'-GCTTGCAAGCTTTATTTAGTTATGACTGATAACACTC                         __________________________________________________________________________

III. Deoxyribonucleoside Triphosphates

The amount of reverse transcription (RT) product formed was monitored bythe incorporation of α³² P dCTP. Therefore, a dNTP minus C stock wasprepared comprising 2 mM dATP, 2 mM dTrP, and 2 mM dGTP. A 330 μl, 1 mMdCTP solution was prepared containing 100 μCi α⁼ p dCTP (New EnglandNuclear). Therefore, approximately 6.6×10⁵ cpm represents 10³ pmolesdCTP incorporated. The dNTP minus C and dCTP solutions were combined toprepare a 5X dNTP stock mix containing 1 mM dATP, 1 mM dTTP, 1 mM dGTP,and 250 μM α³² p dCTP. Alternatively, when no radio-labelledtriphosphate is used, all four dNTPs are included in the reversetranscription reaction at 20.0 μM. For convenience a solution containing2 mM each of dATP, dCTP, dGTP and dTTP in H₂ O, pH 7.0, is prepared as a10X stock solution. Alternatively, reverse transcription/PCR product wasmonitored by agarose gel electrophoresis and ethidium bromide staining.

IV. Buffers

A. Annealing Buffer

The 10X stock annealing buffer was prepared containing 100 mM Tris-HClpH 8.3, 500 mM KCl and 1 mM EDTA.

B. Modified Pol I 10X Buffer

The 10X Pol I buffers were prepared with and without MgCl₂ containing100 mM Tris-HCl, pH 8.3, 500 mM KCl, 10 mM DTT, and 60 mM MgCl₂ ifpresent.

C. Taq Polymerase/Reverse Transcription 10X Buffer (HSB)

The 10X Taq buffer was prepared containing 100 mM Tris-HCl, pH 8.3 and500 mM KCl.

D. 10X Low Salt Buffer (LSB)

The 10X LSB was prepared containing 100 mM Tris-HCl, pH 8.3 and 50 mMKCl.

E. 10X RT Reaction Buffer

The 10X RT buffer was prepared containing 100 mM Tris-HCl (pH 8.3) and900 mM KCl.

F. MoMuLV-RT 10X Buffer

The 10X MoMuLV-RT buffer was prepared as in C, above, with the additionof 60 mM MgCl₂.

G. 10X PCR Buffer

The 10X PCR buffer was prepared containing 100 mM Tris-HCl, pH 8.3, 1MKCl, 18.75 mM MgCl₂, 7.5 mM EGTA, and 50% glycerol (v/v).

H. 10X Taq PCR Buffer

The 10X Taq PCR buffer contained 100 mM Tris-HCL (pH 8.3), 300 mM KCl,and 25 mM MgCl₂.

I. Taq Diluent

Taq dilution buffer was prepared comprising: 25 mM Tris-HCl, pH. 8.8,100 mM KCl, 0.1 mM EDTA, 0.5% Tween-20™, 0.5% Nonidet^(TM) P-40, and 500μg/ul gelatin.

V. Enzymes

A. Reverse Transcriptase (MoMuLV-RT) was obtained from Bethesda ResearchLabs, Bethesda, Maryland at a concentration of 200 u/μl. The enzyme wasdiluted in Taq Diluent with 1/5 concentration of Tween-20™ and Nonidet™P-40 to provide 4, 0.4, 0.04, and 0.004 u/gl preparations.

B. E. coli Pol I was purchased from New England Biolabs at aconcentration of 10 units/μl.

C. Native Taq (94 kDa) was provided by Perkin-Elmer/Cetus at aconcentration of 48 units/μl. The specific activity was approximately240,000 units/mg. Taq diluent was used to reduce the concentration to 10units/μl.

D. rTaq DNA Polymerase, Stoffel Fragment

The Stoffel fragment of Taq polymerase is a truncated form of 94 kDa Taqin which the 32 kDa amino terminal sequence has been deleted. Althoughthe enzyme is 61 kDa in size, it has previously been referred to as 62kDa Taq. The enzyme can be produced in and purified from recombinanthost cells as described in commonly assigned, co-pending Ser. No.143,441, now abandoned, incorporated herein by reference. Stoffelfragment is commercially available from Perkin Elmer Cetus Instrumentsas AmpliTaq™ DNA polymerase, Stoffel Fragment.

E. Tth Polymerase

Native Tth polymerase is commercially available from Finnzyme Co.,Finland, and from Toyobo Co., Japan. Methods for purifying 94 kDa nativeTth DNA polymerase and producing and purifying recombinant 94 kDa Tthare described in commonly assigned, co-pending Ser. No. 455,967, nowabandoned in favor of continuation application U.S. Ser. No. 07/880,478,filed May 6, 1992, and also published as PCT Patent Publication No. WO91/09950, by inventors David Gelfand, Susan Stoffel, and Frances Lawyer,filed Dec. 22, 1989, and incorporated herein by reference. For use inthe present examples, recombinant B (rTth) was purified as describedbelow and is commercially available from Perkin Elmer Cetus Instruments.

Tth was purified from E. Coli. strain DG 116 containing plasmid pLSG33.As described at page 46 of the specification of U.S. patent applicationSer. No. 455,967, now abandoned in favor of continuation applicationU.S. Ser. No. 07/880,478, filed May 6, 1992, and also published as PCTPatent Publication No. WO 91/09950, pLSG33 was prepared by ligating theNdeI-BamHI restriction fragment of pLSG24 into expression vector pDG178. The resulting plasmid is ampicillin resistant and is capable ofexpressing the full-length Tth gene. The seed flask for a 10 literfermentation contains tryprone (20 g/l), yeast extract (10 g/l), NaCl(10 g/l) and 0.005% ampicillin. The seed flask was inoculated fromcolonies from an agar plate, or a frozen glycerol culture stock can beused. The seed is grown to between 0.5 and 1.0 O.D. (A680). The volumeof seed culture inoculated into the fermentation is calculated such thatthe final concentration of bacteria will be 1 mg dry weight/liter. The10 liter growth medium contained 25 mM KH₂ PO₄, 28 mM (NH₄) SO₄, 4 mMsodium citrate, 0.4 mM FeCl₂, 0.04 mM ZnCl₂, 0.03 mM COCl₂, 0.03 mMCuCl₂, and 0.03 mM H₃ BO₃. The following sterile components were added:4 mM MgSO₄, 7.5 g/l glucose, and 20 mg/l thiamine-HCl. The pH wasadjusted to 6.8 with NaOH and controlled during the fermentation byadded NthOH. Glucose was continually added during the fermentation bycoupling to NH₄ OH addition. Foaming was controlled by the addition ofpolypropylene glycol as necessary, as an anti-foaming agent. Dissolvedoxygen concentration was maintained at 40%.

The fermentation was inoculated as described above and the culture wasgrown at 30° C. until an optical density of 21 (A680) was reached. Thetemperature was then raised to 37° C. to induce synthesis of rTthpolymerase. Growth continued for eight hours after induction, and thecells were then harvested by concentration using cross flow filtrationfollowed by centrifugation. The resulting cell paste was frozen at -70°C. and yielded about 500 grams of cell paste. Unless otherwiseindicated, all purification steps were conducted at 4° C.

Approximately 280 grams of frozen (-70° C.) E. coli K12 strain DG116harboring plasmid pLSG33 were warmed overnight to -20° C. To the cellpellet the following reagents were added: 1 volume of 2X TE (100 mMTris-HCl, pH 7.5, 2 mM EDTA), 5 mg/ml leupeptin and 50 mg/ml PMSF. Thefinal concentration of leupeptin was 0.5 μg/ml and for PMSF, 0.625μg/ml. Preferably, betamercaptoethanol (2-Me) is included in TE toprovide a final concentration of 5 mM 2-Me. The mixture was homogenizedat low speed in a blender. All glassware was baked prior to use, andsolutions used in the purification were autoclaved, if possible, priorto use. The cells were lysed by passage twice through a Microfluidizerat 10,000 psi.

The lysate was diluted with 1X TE containing 5 mM 2-Me to a final volumeof 5.5X cell wet weight. Leupeptin was added to 0.5 μg/ml and PMSF wasadded to 0.625 μg/ml. The final volume (Fraction I) was approximately1540 ml.

Ammonium sulfate was gradually added to 0.2 M (26.4 g/l) and the lysatestirred. Upon addition of ammonium sulfate, a precipitate formed whichwas removed prior to the polyethylenimine (PEI) precipitation step,described below. The ammonium sulfate precipitate was removed bycentrifugation of the suspension at 15,000-20,000 xg in a JA-14 rotorfor 20 minutes. The supernatant was decanted and retained. The ammoniumsulfate supernatant was then stirred on a heating plate until thesupernatant reached 75° C. and then was placed in a 77° C. bath and heldthere for 15 minutes with occasional stirring. The supernatant was thencooled in an ice bath to 20° C. and a 10 ml aliquot was removed for PEItitration.

PEI titration and agarose gel electrophoresis were used to determinethat 0.3% PEI (commercially available from BDH as PolyminP) precipitates≧90% of the macromolecular DNA and RNA, i.e., no DNA band was visible onan ethidium bromide stained agarose gel after treatment with PEI. PEIwas added slowly with stirring to 0.3% from a 10% stock solution. ThePEI treated supernatant was centrifuged at 10,000 RPM (17,000 xg) for 20minutes in a JA-14 rotor. The supernatant was decanted and retained. Thevolume (Fraction II) was approximately 1340 ml.

Fraction II was loaded onto a 2.6×13.3 cm (71 ml) phenyl sepharose CL-4B(Pharmacia-LKB) column following equilibration with 6 to 10 columnvolumes of TE containing 0.2 M ammonium sulfate. Fraction II was thenloaded at a linear flow rate of 10 cm/hr. The flow rate was 0.9 ml/min.The column was washed with 3 column volumes of the equilibration bufferand then with 2 column volumes of TE to remove non-Tth DNA polymeraseproteins. The column was then washed with 2 column volumes of 20%ethylene glycol in TE to remove additional contaminating proteins. Therecombinant Tth was eluted with 4 column volumes of 2.5M urea in TEcontaining 20% ethylene glycol. The DNA polymerase containing fractionswere identified by optical absorption (A₂₈₀) and SDS-PAGE according tostandard procedures. Peak fractions were pooled and filtered through a0.2 micron sterile vacuum filtration apparatus. The volume (Fraction IIIwas approximately 195 ml. The resin was equilibrated and recycledaccording to the manufacturer's recommendations.

A 2.6×1.75 cm (93 ml) heparin sepharose C1-6B column (Pharmacia-LKB) wasequilibrated with 6-10 column volumes of 0.15M KCl, 50 mM Tris-HCl, pH7.5, 0.1 mM EDTA and 0.2% Tween 20™, at 1 column volume/hour.Preferably, the buffer contains 5 mM 2-Me. The column was washed with 3column volumes of the equilibration buffer. The Tth polymerase waseluted with a 10 column volume linear gradient of 150-750 mM KClgradient in the same buffer. Fractions (one-tenth column volume) werecollected in sterile tubes and the peak was determined as for FractionIII. Recombinant Tth polymerase eluted with a peak at 0.33M KCl. Thepeak fractions were pooled (Fraction IV, volume 177 ml).

Fraction IV was concentrated to 10 ml on an Amicon YM30 membrane. Forbuffer exchange, diafiltration was done 5 times with 2.5X storage buffer(50 mM Tris-HCl, pH 7.5, 250 mM KCl, 0.25 mM EDTA 2.5 mM DTF and 0.5%Tween-20™) by filling the concentrator to 20 ml and concentrating thevolumes to 10 ml each time. The concentrator was emptied and rinsed with10 ml 2.5X storage buffer which was combined with the concentrate toprovide Fraction V.

Anion exchange chromatography was used to remove residual DNA. Theprocedure was conducted in a biological safety hood and steriletechniques were used. A Waters Sep-Pak plus QMA cartridge with a 0.2micron sterile disposable syringe tip filter unit was equilibrated with30 ml of 2.5X storage buffer using a syringe at a rate of about 5 dropsper second. Using a disposable syringe, Fraction V was passed throughthe cartridge at about 1 drop/second and collected in a sterile tube.The cartridge was flushed with 5 ml of 2.5 ml storage buffer and pusheddry with air. The eluant was diluted 1.5 X with 80% glycerol and storedat -20° C. The resulting final Fraction IV pool (57.5 mls) contained16.1×10⁶ units of activity.

VI. Annealing Procedure

For Examples II, III, and IV the cRNA template and DM156 primer wereannealed at a 20:1 primer: template ratio in 10 mM Tris HCl, pH 8.3, 50mM KCl, 0.1 mM EDTA annealing buffer. To reduce pipeting errors andeliminate tube variations, a master mix was made to provide material for80 reactions.

Annealing was accomplished as follows: the 80 reaction master mix washeated to 85°-87° C. for 4 minutes, then placed in a 70° C. water bathfor 5 minutes, followed by a 60° C. water bath for 5 minutes and finallyallowed to equilibrate at room temperature. The annealed mixture wasthen stored at 4° C. (or alternatively at -20° C.) for future use. Foreach reaction 2.5 gl of master mixture was used containing 0.5 pmol(0.175 μg) cRNA template and 10 pmoles primer. Alternatively, annealingwas accomplished at 70° C. during incubation of the RT reaction.

VII. Determination of α³² PdCTP Incorporation

The amount of isotope incorporated in the reverse transcribed productwas measured by nucleic acid precipitation with trichloroacetic acid(TCA). This method is described in Maniatis et al., 1982, MolecularCloning, Cold Spring Harbor Laboratory, page 473.

EXAMPLE II

Analysis of AW 106 cRNA as a Suitable Template for Reverse Transcription

The annealed AW106 cRNA:DM156 mixture was used as a template for reversetranscription with commercially available reverse transcriptase to testthe suitability of AW106 cRNA as a template.

A 6X reaction mix was prepared containing 1X Pol I RT Buffer plus MgCl₂,1X dNTP Stock, and 3 pmoles template annealed to 60 pmoles primer. Thismix was aliquoted into six tubes. All reactions were set up at 0° C. Ascontrols, one reaction was set up without template but with 10 units ofMoMuLV-RT. Another reaction had no enzyme added. To the remainingreactions, MoMuLV-RT was added as follows: 10 units, 1 unit, 0.1 unit,and 0.01 unit.

All reactions were incubated at 37° C. for 20 minutes. The reactionswere stopped by placing them in a 0° C. water ice/bath and adding EDTAto a final concentration of 10 mM. The α³² PdCTP incorporation wasdetermined by measuring TCA precipitable counts.

The results demonstrated that AW106 cRNA was a suitable template forcDNA synthesis using MoMuLV-RT.

EXAMPLE III

Comparison of E. coli Pol I and Taq Reverse Transcriptase Activities

Using the results of Example II as a standard, E. coli Pol I and nTaqpolymerase were assayed for reverse transcriptase activity using AW106cRNA as a template. As positive controls, DNA templates were substitutedfor the cRNA template in one set of reactions. The results werequantitated as in Example II by measurement of α³² PdCTP incorporation.

A 12X Pol I master mix was prepared containing Pol I RT buffer minusMgCl₂ dNTP stock and 12 units Pol I enzyme. Similarly, a 12X Taq mastermix was prepared containing Taq buffer, dNTP stock, and 12 units ofnative Taq enzyme and Taq diluent. The Pol I and Taq master mixes weredivided to provide six reactions for the RNA template (0.5 pmolescRNA/10 pmole DM156), two reactions for the DNA template (6.25 μg), andtwo control reactions with no template.

For the RNA template MnCl₂ or MgCl₂ was added to the six aliquotscontaining Pol I master mix plus cRNA/DM 156 to achieve saltconcentrations of 0, 0.5 mM MnCl₂, 0.7 mM MnCl₂, 1.0 mM MnCl₂, 2.0 mMMnCl₂, and 6 mM MgCl₂. Six aliquots containing Taq master mix pluscRNA/DM156 were supplemented with MnCl₂ or MgCl₂ so that the final saltconcentration was 0, 0.5 mM MnCl₂, 0.7 mM MnCl₂, 1.0 mM MnCl₂, 2.0 mMMnCl₂, or 2 mM MgCl₂.

For the DNA template two aliquots were removed from the Pol I mix, andsalt was added as to provide a final concentration of 0.7 mM MnCl₂ forone reaction and 6 mM MgCl₂ for the other. Two aliquots were removedfrom the Taq mix, and salt was added to provide a final concentration of0.7 mM MnCl₂ for one reaction and 2 mM MgCl₂ for the other.

As negative controls, two reaction mixes were prepared for each of Pol Iand Taq which lacked a template. These reactions were assembled usingaliquots of the 12X Pol I and 12X Taq master mixes, and 1X annealingbuffer was added in place of a template. For Pol I, salt was added toprovide either 0.7 mM MnCl₂ or 6 mM MgCl₂. For Taq, salt was added toprovide either 0.7 mM MnCl₂ or 2 mM MgCl₂.

All reactions were mixed on ice and then incubated at 37° C. for Pol Ior at 65° C. for Taq. After 20 minutes the reactions were chilled on ice(0° C) and EDTA was added to each reaction to a 10 mM finalconcentration. A sample was removed from each reaction to measure α³²PdCTP incorporation.

The results of this experiment are shown in Table I. All values shownare corrected for background.

                  TABLE I                                                         ______________________________________                                                  MoMuLV-RT  nTaq    E. coli Pol I                                              (cpm)      (cpm)   (cpm)                                            ______________________________________                                        minus template +                                                                          90           --      --                                           10 units enzyme                                                               minus enzyme +                                                                            14           --      --                                           template                                                                      10 units enzyme +                                                                         7,825        --      --                                           template                                                                      1 unit enzyme +                                                                           3,263        --      --                                           template                                                                      .1 unit enzyme +                                                                          924          --      --                                           template                                                                      .01 unit enzyme +                                                                         170          --      --                                           template                                                                      RNA template +                                                                            --           9       0                                            0 mM MnCl.sub.2                                                               RNA template +                                                                            --           256     7,561                                        .5 mM MnCl.sub.2                                                              RNA template +                                                                            --           3,088   6,666                                        .7 mM MnCl.sub.2                                                              RNA template +                                                                            --           3,977   7,508                                        1 mM MnCl.sub.2                                                               RNA template +                                                                            --           2,696   1,558                                        2 mM MnCl.sub.2                                                               RNA template +                                                                            --           73      --                                           2 mM MgCl.sub.2                                                               RNA template +                                                                            --           --      760                                          6 mM MgCl.sub.2                                                               minus template +                                                                          --           --      31                                           6 mM MgCl.sub.2                                                               minus template +                                                                          --           5       28                                           .7 mM MnCl.sub.2                                                              minus template +                                                                          --           3       --                                           2 mM MgCl.sub.2                                                               DNA template +                                                                            --           194,199 203,861                                      .7 mM MnCl.sub.2                                                              DNA template +                                                                            --           --      271,595                                      6 mM MgCl.sub.2                                                               DNA template +                                                                            --           209,559 --                                           2 mM MgCl.sub.2                                                               ______________________________________                                    

The data presented in Table I is presented graphically in FIG. 1.

This experiment demonstrates that Taq has reverse transcriptaseactivity. One unit of Taq is equivalent to 1 unit of MoMuLV reversetranscriptase by the amount of α³² PdCTP incorporated into a DNAtranscript from an RNA template. E. coli Pol I also shows reversetranscriptase activity. Because Taq reactions were done at 65° C. ratherthan 37° C., product specificity is enhanced in the Taq reactioncompared to either the Pol I or MoMuLV reverse transcriptase.

EXAMPLE IV

Comparison of Reverse Transcriptase Activity in 94 kDa rTaq, 62 kDa rTaqand Tth Polymerase

In order to determine whether the reverse transcriptase activityobserved in Example III was common to other thermostable polymerases,the reverse transcription activity of 94 kDa Taq polymerase, Stoffelfragment (previously referred to as 62 kDa Taq), and native Tth werecompared. Both forms of Taq were produced by recombinant means.

A 2 μM dilution of 94 kDa Taq was prepared, assuming 94 μg/nmole, from a23.4 μM stock solution. A dilution of the Stoffel fragment was similarlyprepared using Taq diluent.

Both the 94 kDa and Stoffel fragment dilutions contained 0.36pmoles/0.18 μl. Tth polymerase was purified as a 27 unit/gl solutionwith a specific activity of 80,000 units/mg. Therefore, 0.1 μl contained0.36 pmole (2.7 units of enzyme). Reaction were set up with a final saltconcentration of 60 mM KCl (HSB) or 15 mM KCl (LSB).

At 0° C. three 15X master mixes were prepared containing dNTP stock,enzyme diluent, and 5.4 pmoles enzyme (Tth, 94 kDa Taq, or StoffelFragment). From each 15X master mix six aliquots were combined witheither HSB or LSB providing six reaction mixes for each of Tth/HSB,Tth/LSB, 94 kDa Taq/HSB, 94 kDa Taq/LSB, Stoffel Fragment/HSB andStoffel Fragment/LSB.

For each of the six reaction mixes two separate aliquots were removed totubes containing 1X annealing buffer for the minus template plus enzymecontrol reactions.

To the remaining five reactions worth of reaction mix, cRNA/DM156annealed mix (3 pmoles template and 60 pmoles primer) was added. Fromeach of the six series, four aliquots were removed to individual tubes.While still at 0° C., MnCl₂ was added to provide the final saltconcentration show in Table II.

To determine background levels, minus-enzyme, minus-template controlswere prepared containing 1X dNTP stock, 1X Annealing buffer, and 0.1XTaq diluent. The salts were adjusted as follows: HSB and a final MnCl₂concentration of 0.6 mM or 1.2 mM, and LSB and a final MnCl₂concentration of 0.6 mM or 1.2 mM.

All reaction mixes were incubated at 65° C. for 15 minutes. The tubeswere then quenched in an ice bath and EDTA was added to each tube to a10 mM final concentration. The amount of α³² PdCTP incorporation wasdetermined by TCA precipitation. The results are shown in Table II.

                                      TABLE II                                    __________________________________________________________________________                      Stoffel Fragment                                                              94 kDA rTaq                                                                          (62 kDa Taq)                                                                         Tth Pol                                                         cpm                                                                              pMoles                                                                            cpm                                                                              pMoles                                                                            cpm                                                                              pMoles                                     __________________________________________________________________________    HSB                                                                           Minus template + 0.6 mM MnCl.sub.2                                                              0  --  0  --  10 --                                         Minus template + 1.2 mM MnCl.sub.2                                                              0  --  0  --  6  --                                         Plus template + 0.6 mM MnCl.sub.2                                                               517                                                                              5.04                                                                              34 0.332                                                                             1244                                                                             12.14                                      Plus template + 0.8 mM MnCl.sub.2                                                               918                                                                              8.96                                                                              340                                                                              3.32                                                                              1981                                                                             19.33                                      Plus template + 1.0 mM MnCl.sub.2                                                               1315                                                                             12.83                                                                             521                                                                              5.08                                                                              2178                                                                             21.25                                      Plus template + 1.2 mM MnCl.sub.2                                                               1305                                                                             12.73                                                                             609                                                                              5.9 2369                                                                             23.11                                      LSB                                                                           Minus template + 0.6 mM MnCl.sub.2                                                              7  --  0  --  234                                                                              2.28                                       Minus template + 1.2 mM MnCl.sub.2                                                              18 --  0  --  2  --                                         Plus template + 0.6 mM MnCl.sub.2                                                               276                                                                              2.69                                                                              81 0.79                                                                              618                                                                              6.03                                       Plus template + 0.8 mM MnCl.sub.2                                                               1115                                                                             10.88                                                                             468                                                                              4.57                                                                              2263                                                                             23.06                                      Plus template + 1.0 mM MnCl.sub.2                                                               1349                                                                             13.16                                                                             1068                                                                             10.46                                                                             2239                                                                             21.85                                      Plus template + 1.2 mM MnCl.sub.2                                                               1061                                                                             10.35                                                                             898                                                                              8.76                                                                              2051                                                                             20.01                                      Controls                                                                      Minus Enzyme, Minus Template Reactions                                                             cpm                                                      60 mM KCl .6 mM MnCl.sub.2                                                                         19                                                       60 mM KCl 1.2 mM MnCl.sub.2                                                                        46                                                       15 mM KCl .6 mM MnCl.sub.2                                                                         11                                                       15 mM KCl 1.2 mM MnCl.sub.2                                                                        25                                                       __________________________________________________________________________

Input ³² p for each reaction was 1.23×10⁶ cpm. All numbers werecorrected for average background of 37 cpm. The numbers reflect cpmincorporated per 12.5 μl of each reaction. Total pmoles of incorporationwas calculated based on 984 cpm/pmole determined by counting 32p from anα³² P-dCTP stock solution.

These results are presented graphically in FIG. 2 and demonstrate thatall thermostable DNA polymerases tested contain reverse transcriptaseactivity.

EXAMPLE V

Procedure for High Temperature Reverse Transcription/Amplification

Examples III and IV demonstrate the ability of thermostable DNApolymerases to use an RNA template and produce a cDNA molecule, at anelevated temperature. In this experiment, the reverse transcriptasereaction was coupled to cDNA amplification by PCR. Recombinant Tth wasused as the thermostable polymerase for both the reverse transcriptasereaction and PCR. The cRNA template, prepared from plasmid pAW109 isdescribed in Example 1(A). This embodiment of the invention excludes thepre-annealing step described in Example II and used in Examples III andIV.

The components for the RT reaction were combined at room temperature, inthe following order: 9.4 gl, H₂ O; 2 μl, 10X RT Reaction Buffer (100 mMTris HCl (pH 8.3), 900 mM KCl); 2 μl, 10 mM MnCl₂ ; 1.6 μl, dNTPsolution (2.5 mM each dATP, dCTP, dGTP, dTTP in H₂ O at pH 7.0); and 2μl, rTth DNA polymerase (2.5 units/μl in 1X enzyme storage buffercontaining 20 mM Tris-HCl [pH 7.5], 100 mM KCl, 0.1 mM EDTA 1 mM DTT,0.2% Tween 20™ [Pierce Surfactamps] and 50% glycerol [v/v]). Althoughthe indicated volumes shown are intended as per reaction, forconsistency and to avoid pipeting errors, the RT reaction mix wasprepared as a 25X master mix. The 25X reaction master mix contained 425μl (17 μl/Reaction).

RT-Primer mixes were prepared each as follows. 187 μl of RT mix wasremoved from the 25X RT master mix and combined with a "downstream"primer. This amount was sufficient for 11 RT reactions. Two RT-Primermixes were prepared each containing 187 μl RT reaction mix and 11 μl (1μl per reaction) of either 15 μM DM152 (in water); or 15 μM TM01 (inwater). Aliquotes comprising 18 μl of the DM152 RT-Primer mix wereremoved into tubes labeled 1-8. Similarly, 18 μl aliquotes of the TM01RT-Primer mix were removed into tubes numbered 9-16.

Template AW 109 cRNA was prepared as described in Example I, diluted,and added as a 2 μl template solution in TE (10 mM Tris-HCl, 1 mM EDTA),as shown below. The template solution contained 30 ng/μl rRNA ascarrier.

    ______________________________________                                        Tube Number  Copies of AW109 cRNA                                             ______________________________________                                        1, 9         10.sup.8    (-RT reaction)                                       2, 10        10.sup.8                                                         3, 11        10.sup.6                                                         4, 12        10.sup.4                                                         5, 13        10.sup.3                                                         6, 14        500                                                              7, 15        100                                                              8, 16        0                                                                ______________________________________                                    

Reaction tubes 2-8 and 10-16 were incubated at 70° C. for 2.5 minutesfor DM152 and 7.5 minutes for TM01 samples. Tubes 1 and 9 were kept onice as RT reaction negative controls to detect the presence ofcontaminating plasmid DNA that could later serve as a PCR template.After incubation at 70° C., the reactions were stopped by placing thetubes on ice.

The PCR assay mix was prepared at room temperature as a 19X master mix.The volumes shown are intended as volume per reaction: 71 μl H₂ O; 8 μl10X PCR Reaction Buffer (100 mM Tris-HCl [pH 8.3]; 1M KCl, 18.75 mMMgCl₂ ; 7.5 mM EGTA; 50% glycerol [v/v]) and 1 μl, 15 μM DM151 (the "PCRupstream primer"). The total volume was 80 gl per reaction.

The PCR amplification was initiated by adding the 80 μl PCR assaymixture to the 20 μl reverse transcriptase reaction. A mineral oiloverlay (75 μl) was added to prevent evaporation and the mix was thenspun in a microcentrifuge for approximately 20 seconds to separate theoil layer from the reaction mix. PCR was conducted using a Perkin ElmerCetus Instruments Thermal Cycler and four linked files as follows:

File 1--Step Cycle 2 minute at 95° C. for 1 cycle

File 2--Step Cycle 1 minute at 95° C. and 1 minute at 60° C. for 35cycles

File 3--Step Cycle 7 minute at 60° C. for 1 cycle

File 4--Soak 4° C.

Following PCR, 5 μl aliquots were removed from each sample and combinedwith 5 μl sample dye (30% w/v sucrose, 0.1% w/v bromophenol blue, 10 mMEDTA) analyzed on an agarose-gel (2% Nu Sieve® GTG agarose [FMC], 1%Seakem® ME agarose [FMC]). Following electrophoresis, the gel wasstained with ethidium bromide and photographed (see FIG. 3). All productlengths were determined relative to a 1 kb BRL molecular weight standard(lane not shown). In the figure, the lane numbers correspond to tubenumbers. The expected product, 308 bp in length, was visible at 100copies AW109 cRNA per reaction. When TM01 was used to produce a 730 basepair transcription/amplification product, the correct size band wasvisible at 100 molecules of template per reaction. No PCR product wasdetected in the negative control reactions.

EXAMPLE VI

Coupled Reverse Transcription/Amplification Using Total Cellular RNA

The K562 cell line was used as a source of total cellular RNA. The RNAwas purified as described in Example I. The purpose of this experimentwas to examine the sensitivity of the coupled RT/PCR procedure using anaturally occurring heterogenous RNA composition. Generally, it can beassumed that 250 ng of total RNA per reaction represents approximately25,000 cells. Each cell contains approximately 1-10 copies of IL- 1αmRNA. Therefore, 250 ng of K562 total RNA contains roughly 25,000 to250,000 copies of IL- 1α target mRNA. Thus, the specificity and amountof PCR product can be compared to the specificity and amount of productmade using the synthetic cRNA template in Example V.

The reaction conditions were as described in Example V using DM151 andDM152 with a few minor changes described below. Because only onedownstream primer was used in this experiment, DM 152 was added directlyto the RT reaction mix. A 10X RT reaction master mix was preparedcontaining, for each reaction, 9 μl H₂ O; 2 μl 10X RT Reaction Buffer; 2μl 10 mM MnCl₂ ; 2 gl dNTP (2 μl each dATP, dCTP, dGTP, and dTYP in H₂O, pH 7.0) and 1 μl DM152 (15 μM in water). The RT master mix wasprepared at room temperature and 16 gl aliquots were dispensed intotubes numbered 1-9 containing RNA as shown below.

    ______________________________________                                        Tube Number  K562       Total RNA                                             ______________________________________                                        1            250        ng (-RT control)                                      2            250        ng                                                    3            50         ng                                                    4            10         ng                                                    5            2          ng                                                    6            0.4        ng                                                    7            0.08       ng                                                    8            0          ng                                                    ______________________________________                                    

All template solutions were in TE (10 mM Tris-HCl, 1 mM EDTA). Two gl oftemplate solution and 2 gl of Tth polymerase (2.5 units/μl in 1X enzymestorage buffer) were added to each tube.

All samples were incubated at 70° C. for 2.5 minutes with the exceptionof tube 1 which was kept on ice as a negative RT control to test for thepresence of contaminating DNA that might serve later as a PCR template.The reactions were stopped by placing them on ice.

The PCR assay mix was prepared, and the reaction was carried out exactlyas described in Example V. The RT/PCR results were analyzed as inExample V, and the results are shown in FIG. 4. A PCR product band wasvisible in lanes 2-7. The results, shown in FIG. 4, demonstrate that aslittle as 80 picograms of total cellular RNA (corresponding to 8-80 cellequivalents of RNA) serves as an excellent template for specific andefficient high temperature reverse transcription and amplificationaccording to the methods of the present invention.

EXAMPLE VII

Procedure for High Temperature Reverse Transcription/AmplificationWherein the Polymerase is Exchanged

Example IV suggests that Tth polymerase may be superior to Taqpolymerase for preparing cDNA. However, Taq polymerase is frequentlyused in PCR. Therefore, a procedure was developed wherein Tth polymerasecatalyzes the RT reaction and Taq polymerase catalyzes PCR. Thefollowing procedure is suitable when the two reactions are catalyzed bydifferent thermostable DNA polymerases or when the amount of polymerasein the RT reaction is decreased for PCR.

As an illustration, the following experiment was carried out. Generally,the RT reaction was carded out as in Example V, however, for half of thereaction tubes, the Tth was heat killed and replaced with Taq for PCR.

The specific protocol was as follows. The RT master mix was prepared,with DM152, exactly as described in Example VI. The RT master mix wasmade up as a 9X mix. Sixteen μl aliquots were removed into tubesnumbered 1-8 containing AW109 cRNA or pAW 109 DNA as shown below.

Template solutions were all prepared as 2 gl samples in TE as in ExampleV. Two μl of rTth was added to each of tubes 1-8 (2.5 units/μl in 1Xenzyme storage buffer) and the RT reactions were incubated at 70° C. for2.5 minutes, with the exception of tubes 1, 2, 5, and 6. These tubeswere kept on ice as RT reaction negative controls. The reactions werestopped by placing the tubes on ice. The table below summarizes thereaction conditions for each tube.

    ______________________________________                                        Lane   Sample          Reaction Enzyme                                        ______________________________________                                        1      10.sup.4 copies DNA                                                                           -RT      -Taq                                          2      10.sup.4 copies cRNA                                                                          -RT      -Taq                                          3      10.sup.4 copies cRNA                                                                          +RT      -Taq                                          4      --              +RT      -Taq                                          5      10.sup.4 copies DNA                                                                           -RT      +Taq                                          6      10.sup.4 copies cRNA                                                                          -RT      +Taq                                          7      10.sup.4 copies cRNA                                                                          +RT      +Taq                                          8      --              +RT      +Taq                                          ______________________________________                                    

At room temperature, two PCR master mixes were prepared. PCR minus Taqcontained, per reaction, 7 1 μl H₂ O, 8 gl, 10X PCR reaction buffer (100mM Tris-HCl, pH 8.3; 1M KCl; 18.75 mM MgCl₂, 7.5 mM EGTA, 50% glycerol[w/v]) and 1 μl DM151 (15 μM in water). The PCR minus Taq mix wasprepared as a 5X solution. A PCR plus Taq master mix was also preparedas a 5X solution containing, per reaction, 68.5 μl H₂ O; 8 μl 10XTaq-PCR reaction buffer (100 mM Tris-HCl, pH 8.3; 300 mM KCl; 25 mMMgCl₂), 1 μl DM151, and 0.5 μl AmpliTaq™ (5 units/μl).

Eighty gl of PCR minus Taq master mix were added to tubes 1-4. EGTA (2μl of 30 mM stock) was added to tubes 5-8. Mineral oil was then added toall tubes (75 μl/tube). Tubes 5-8 were heated to 99° C. for 4 minutes,and 78 μl of PCR plus Taq reaction mix was added to those tubes only(below the oil level). All tubes were spun in a microcentrifuge forapproximately 20 seconds and incubated in a thermocycler using the fourlinked files described in Example V. The RT/PCR amplifications wereanalyzed by electrophoresis as described in Example V, and the gel wasphotographed (FIG. V). FIG. V demonstrates that replacement of rTth withAmpliTaq™ in the PCR step does not effect product yield.

EXAMPLE VIII

Comparison of Taq Polymerase and Tth Polymerase in a Coupled RT/PCRReaction

The use of the cRNA standard described in Example VII facilitates directanalysis of the effect of experimental conditions on RT/PCR efficiency,because the number of target molecules present in the reaction mix isknown. Specifically, the efficiency of Tth and Taq polymerases in acoupled RT/PCR reaction were compared. AmpliTaq® DNA polymerase (330units/μl) and rTth (1697 units/μl) were provided by Perkin Elmer CetusInstruments. The enzymes were diluted to 2.5 units/gl in storage buffer(20 mM Tris-HCl, [pH 7.5], 100 mM KCl, 50% glycerol [v/v], 0.1 mM EDTA,1 mM dithiothreitol and 0.2% Tween® 20).

RT reactions (20 gl) contained 10 mM Tris-HCl, pH 8.3; 90 mM KCl (40 mMfor reactions containing Taq); 1.0 mM MnCl₂, 200 μM each of dATP, dCTP,dGTP, and dTTP; 15 pmol of DM152 and 5 units of rTth or Taq and 10⁶,10⁵, or 10⁴ copies of pAW109 cRNA. The six reactions were overlaid with75 μl mineral oil and incubated for 15 minutes 70° C.

Following the RT reaction, 80 μl of a solution containing 10 mMTris-HCl, pH 8.3, 100 mM KCl, (50 mM for reactions containing Taq) 1.88mM MgCl₂, 0.75 mM EGTA; 5% glycerol [v/v] and 15 pmol of primer DM151was added. The samples (100 μl) were then amplified in a Perkin ElmerCetus Instruments Thermal Cycler as follows: 2' at 95° C. for 1 cycle;1' at 95° C. and 1' at 60° C. for 35 cycles; and 7' at 60° for 1 cycle.Aliquots (5 μl) of the PCR amplifications were analyzed byelectrophoresis on 2% (w/v) NuSeive® 1% (w/v) Seakem® agarose stainedwith ethidium bromide.

Results

The rTth polymerase generated a 308 bp product visualized by ethidiumbromide stained gel electrophoresis starting with 10⁴ copies of targetcRNA. Product was not observed for the Taq polymerase at 10⁴ or 10⁵copies of target, although lower limits of detection would be expectedif hybridization techniques were used rather than ethidium bromidestaining. These results demonstrated that under similar reactionconditions the Tth polymerase provides approximately 100-fold greatersensitivity than the analogous Taq polymerase in a coupled reversetranscription PCR amplification.

EXAMPLE IX

Preferred Non-Homogeneous Reverse Transcription/PCR Protocol

A. Reverse Transcription Reaction

In a 0.5 ml polypropylene microcentrifuge tube combine 9.4 mi steriledistilled water; 2 μl 10X rTth RT buffer; 2 μl MnCl₂ (10 mM); 0.4 μl ofeach of dGTP, dATP, dTTP, and dCTP (each at 10 mM); 2 μl rTth polymerase2.5 U/μl; 1 μl of primer DM152 (15 μM) (or an alternative "downstream"primer); and 2 μl positive control RNA or experimental samplecontaining<250 ng total RNA.

In this embodiment, the positive control RNA serves a template forDM152. The control RNA concentration is preferably ˜10⁴ copies/20 μl.For example, the control RNA may be RNA transcribed from pAW109 in 30μg/ml E. coli rRNA in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA and 10 mM NaCl.

The total reverse transcription reaction volume should be 20 μl persample.

To reduce evaporation or refluxing, overlay the mix with 50-100 μlmineral oil.

Incubate the tubes in a Perkin-Elmer Cetus DNA Thermal Cycler using asoak file at 70° C. for 5-15 minutes. Stop the reaction by placing thetubes on ice until needed.

B. PCR Reaction

For each sample prepare a minimum of 80 μl of PCR master mix as follows:8 μl, 10X chelating buffer, 6-10 μl 25 mM MgCl₂, 1 μl primer DM151 (15μM) or experimental "upstream" primer and sterile distilled water. Anycombination of water, MgCl₂ and "upstream" primer volumes can be used aslong as the total volume of the master mix equals 80 gl per sample.

The optimal MgCl₂ concentration may vary, depending on the total dNTPconcentration, and the primer and template used. In most cases a finalconcentration of MgCl₂ in the range of 1.5-2.5 mM in the reaction mixwill provide excellent results. If the template used is the positivecontrol pAW 109 RNA, 6 μl (1.5 mM) of MgCl₂ is preferred.

Dispense 80 μl of the PCR master mix into each reverse transcriptionreaction tube. Change piper tips between additions to avoid samplecarryover. Centrifuge the tubes for ˜30 seconds in a microcentrifuge.

For amplification of the pAW109 RNA positive control, the Perkin ElmerCetus DNA Thermal Cycler is programmed for four linked files as follows:

Step Cycle: 2 minutes at 95° C. for 1 cycle

Step Cycle: 1 minute at 95° C. and 1 minute at 60° C. for 35 cycles

Step Cycle: 7 minutes at 60° C. for 1 cycle

Soak: 4° C.

The PCR amplified samples can be stored frozen until subsequentanalysis.

The selection of 60° C. for the anneal-extend temperature is optimal foramplification of the positive control cDNA. It may be necessary to loweror raise the anneal-extend temperature for other primer-template pairs.Higher anneal-extend temperatures generally result in a specific product(see Saiki et al., 1988, Science 239:487-491). The optimum can bedetermined empirically by testing at 5° C., or smaller, increments untilthe maximum in specificity and product yield is reached.

The optimal magnesium chloride concentration for PCR amplification canbe determined empirically by testing concentrations from 1.5 to 2.5 mMmagnesium chloride for each primer set. Too little or too much magnesiumchloride may effect amplification efficiency. It may be preferable toadjust the magnesium chloride concentration in parallel with substantialchanges in the concentration of sample RNA, dNTPs, cDNA, and DNA.

For templates known to contain a high amount of secondary structure, a"hot start" protocol may be preferred. Two reaction mixes for thereverse transcription reaction are prepared. Mix A: 9.4 μl steriledistilled water; 2 μl 10X rTth reverse transcriptase buffer; 1 μl"downstream primer;" 2 μl sample RNA (<250 ng of total RNA). Mix B: 2μl, 10 mM MnCl₂ solution; 0.4 μl dGTP; 0.4 μl dATP; 0.4 μl dCTP; 0.4 μldTrP; 2 μl rTth DNA polymerase.

Prepare both reaction mixes at room temperature. Incubate Mix A for 5minutes at 70° C., add reaction Mix B (while reaction Mix A is still at70° C.) and incubate for 5 to 15 minutes at 70° C. as described above inthe section entitled "Reverse Transcription Reaction." Run the PCRreaction as described above.

C. Reagents

    ______________________________________                                        The preferred protocol utilizes the following reagents:                       ______________________________________                                        rTth DNA polymerase                                                                          2.5 Units/μl                                                Primer DM152   15 μM                                                       Primer DM151   15 μM                                                       Positive Control RNA                                                                         5 × 10.sup.3 copies/μl                                dATP           10 mM                                                          dGTP           10 mM                                                          dCTP           10 mM                                                          dTTP           10 mM                                                          10X rTth Reverse                                                                             100 mM Tris-HCl                                                Transcriptase RT Buffer:                                                                     pH 8.3, 900 mM KCl                                             10X Chelating Buffer:                                                                        50% glycerol (v/v)                                                            100 mM Tris HCl, pH 8.3, 1 M KCl,                                             7.5 mM EGTA, 0.5% Tween 20                                     MnCl.sub.2 Solution                                                                          10 mM                                                          MgCl.sub.2 Solution                                                                          25 mM                                                          ______________________________________                                    

These components may be assembled as a kit for high temperature reversetranscription. Variations to the kit are within the scope of thedisclosed invention. For example, MnCl₂ may be included in the reversetranscriptase buffer and MgCl₂ may be included in the Chelating buffer.However, for optimization of the reactions MnCl₂ and MgCl₂ are providedas separate reagents. The use of a positive control, while notessential, is preferred in a commercial embodiment of the invention.

EXAMPLE X Homogeneous RT/PCR Assay

This method provides a procedure for a two-step, single addition reversetranscription/PCR reaction. ATC 9600 thermocycler (Perkin Elmer CetusInstruments) was used and the instrument was turned on, to preheat thecover, prior to preparing the reaction mixture. In a 0.2 ml MicroAmp®tube (Perkin Elmer Cetus Instruments), each contained 6.4 μl steriledistilled H₂ O; 2 μl 10X RT buffer (100 mM Tris-HCl, pH 8.3; 900 mMKCl); 1.6 μl of 10 mM MnCl₂ ; 2 μl of 10X dNTP-T (2 mM each dATP, dCTP,dGTP in H₂ O pH 7.0); 2 μl of 2 μl dTTP; 1 μl of primer DM152 (15 μM); 1μl of primer DM151 (15 μM); and 2 μl rTth (2.5 U/μl). A 20X reactionmixture was made up (360 μl total volume) and 18 μl mixture wasaliquoted into 16 tubes containing template as described below. Thetemplate used was AW109 cRNA. Tube Nos. 1-3 and 9-11 contained 10⁴copies of template in 2 μl. Tube Nos. 4-6 and 12-14 each contained 10²copies in 2 μl. Tube Nos. 7, 8, 15, and 16 contained only 2 μl of 30ng/μl rRNA as a negative control.

Tube Nos. 1-8 were kept on ice during the RT reaction as -RT controls.Tube Nos. 9-16 were placed in a TC9600 thermocycler and heated for 1cycle at 70° C. for 15' and then heated to 95° C. while Tube Nos. 1-8were placed in the thermocycler for the PCR step. All tubes were cycledas follows:

75 seconds 95° C. 1 cycle

30 seconds 95° C., 20 seconds 60° C. for 35 cycles

2 minutes 60° C. 1 cycle

Results

Five μl of each reaction was then analyzed on a 2% NuSieve 1% agarosegel, strained with ethidium bromide and photographed. No product of thepredicted size was visible in the -RT controls (Tube Nos. 1-8) or the"no target controls" (Tube Nos. 15 and 16). Product of the expected sizewas readily visible in lanes 9-11 (10⁴ copies of target) and alsopresent in lanes 12-14, although, expectedly, with less intensity.

EXAMPLE XI

Utilization of dUTP and Uracil-N-Glycosylase (UNG) as a PCR CarryoverPrevention During High Temperature Reverse Transcription andAmplification

This example illustrates the incorporation of an unconventionalnucleotide to minimize carryover contamination. The reaction mix wastreated with UNG prior to reverse transcription to degrade contaminatingproducts from previous assays containing the same unconventionalnucleotide. UNG treatment is as follows: 0.5 units UNG (Perkin ElmerCetus Instruments) per 20 μl RT reaction. The reaction was incubated for10 minutes at room temperature followed by heating at 70° C. for 15minutes to denature the glycosylase and allow for reverse transcription.The experiment also demonstrates MnCl₂ concentration titration fordetermining the optimum concentration for the particular target,primers, and reaction conditions shown. The cDNA is then amplified by aPCR.

An 8X RT reaction mixture was prepared that contained: 48 μl sterileDEPC-treated distilled water; 16 μl 10X RT Buffer (100 mM Tris-HCl, pH8.3; 900 mM KCl); 16 μl of a dNTP mix containing 2 mM each of dATP,dCTP, dGTP, and dUTP; 16 μl each of DM152 (1.5 μM) and DM151 (1.5 μM);16 μl of AW109 cRNA template (5×10³ copies/μl); and 16 μl of rTth (2.5units/gl). The final volume was 144 μl (18 μl/reaction). A 7X PCR mastermixture was prepared that contained: 297 μl sterile DEPC-treateddistilled water; 56 μl 10X PCR buffer (100 mM Tris-HCl, pH 8.3; 1M KCl;7.5 mM EGTA; 50% glycerol [v/v]); 140 μl 10 mM MgCl₂ ; 56 μl dNTP mixcontaining 2 mM each of dATP, dCTP, dGTP, dUTP; 5.6 μl of each of DM152and DM151 (15 μM). The final volume was 560 μl, 80 μl per reaction.

Eighteen μl of the RT mix was aliquoted into six sterile microcentrifugetubes and MnCl₂ added in a 2 μl volume to provide a final MnCl₂concentration as follows: Tube Nos. 1 and 2 (1.2 mM MnCl₂); Tube Nos. 3and 4 (1.0 mM MnCl₂); and Tube Nos. 5 and 6 (0.8 mM MnCl2). A mineraloil overlay (75 gl) was added to each tube and the reactions wereincubated at 70° C. for 15' in a water bath. Following the 70° C.incubation, 80 μl of the PCR master mix was added to each. The reactiontubes were thermocycled as follows: 2 minutes at 95° C. for 1 cycle; Iminute at 95° C. and 1 minute at 60° C. for 35 cycles; 7 minutes at 60°C. for 1 cycle; and soak at 4° C.

Results

Five gl of each reaction mix was electrophoresed on a 2% NuSieve 1%agarose gel. The gel was stained and photographed. PCR product of theexpected size was clearly visible in samples from all three MnCl₂concentrations. The product yield increased with increasing MnCl₂concentration.

EXAMPLE XII

Procedure for Sterilization of a Homogeneous RT/PCR Assay

This example illustrates a method for sterilization of a homogeneousRT/PCR reaction contaminated with nucleic acids generated from aprevious reaction. The reaction mix is treated with UNG prior to reversetranscription.

The unconventional nucleotide, dUTP, is incorporated during the RT/PCRreaction. Consequently, any product DNA present as a contaminant insubsequent reactions can be hydrolyzed using UNG.

In a 0.2 ml MicroAmp® tube combine 5.5 μl sterile distilled water; 2 μl10X RT buffer (100 mM Tris-HCl, pH 8.3; 900 mM KCl); 2 μl of 8 mM MnCl₂; 2 μl dNTP mix containing 2 mM each of dATP, dCTP, dGTP, and dUTP; 2 μleach of DM152 (1.5 μM) and DM151 (1.5 μM); 2 μl of AW109 cRNA template(5×10³ copies/μl); 0.5 μl UNG (1 unit/μl); and 2 μl of rTth (2.5units/μl). The reaction is incubated for 10 minutes at room temperatureand subsequently heated at 70° C. for 15 minutes to denature theglycosylase prior to reverse transcription. The cDNA is then amplifiedby a PCR.

In this example, the positive control RNA serves as a template for DM152and DM151 is the upstream primer. The total reaction volume is 20μl/sample. Incubate the tubes in a Thermal Cycler (for example, PECI TC9600) as follows:

70° C. for 15 minutes for 1 cycle

95° C. for 15 seconds and 60° C. for 20 seconds for 2 cycles

90° C. for 15 seconds and 60° C. for 20 seconds for 33 cycles

60° C. for 4 minutes for 1 cycle

The optimal manganese concentration may vary depending on the particularsample, target, primers, and the dNTP concentration in the reactionmixture.

Deposition of Cultures

The cultures were deposited in the Cetus Master Culture Collection(CMCC), 1400 Fifty-Third Street, Emeryville, Calif. 94608, USA, andaccepted by the American Type Culture Collection (ATCC), 12301 ParklawnDrive, Rockville, Md., USA. The CMCC and ATCC accession numbers and ATCCdeposit dates for the deposited samples are given below:

    ______________________________________                                        Culture        ATCC No.  Deposit Date                                         ______________________________________                                        pBSM:Tth10     68195     12/21/89                                             pAW109         68152     10/27/89                                             ______________________________________                                    

These deposits were made under the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purpose of(Budapest Treaty). This assures maintenance of a viable culture for 30years from date of deposit. The deposits will be made available by ATCCunder the terms of the Budapest treaty, and subject to an agreementbetween applicants and ATCC which assures permanent and unrestrictedavailability upon issuance of the pertinent U.S. patent. The Assigneeherein agrees that if the culture on deposit should die or be lost ordestroyed when cultivated under suitable conditions, it will be promptlyreplaced upon notification with a viable specimen of the same culture.Availability of the deposits is not to be construed as a license topractice the invention in contravention of the rights granted under theauthority of any government in accordance with its patent laws.

These deposits were made for the convenience of the relevant public anddo not constitute an admission that a written description would not besufficient to permit practice of the invention or an intention to limitthe invention to these specific constructs. Set forth hereinabove is acomplete written description enabling a practitioner of ordinary skillto duplicate the constructs deposited and to construct alternative formsof DNA, or organisms containing it, which permit practice of theinvention as claimed.

The invention has been described in detail, but it will be understoodthat variations and modifications can be effected within the spirit andscope of the following claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 12                                                 (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other Nucleic Acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      GGCATATGGCTAGACTATTTCTTTTTG27                                                 (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other Nucleic Acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      AGGTTCCGATGAAGTCTGTAGGTGATGTCTG31                                             (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other Nucleic Acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      CTACAGACTTCATCGGAACCTCCTTAAGCG30                                              (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other Nucleic Acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      CCAACCCGCCTCGGCCACGAAGG23                                                     (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other Nucleic Acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      AGTTGGAGGACATCAAGCAGCCATGCAAAT30                                              (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other Nucleic Acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      TGCTATGTCAGTTCCCCTTGGTTCTCT27                                                 (2) INFORMATION FOR SEQ ID NO: 7:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other Nucleic Acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                      ATAATCCACCTATCCCAGTAGGAGAAAT28                                                (2) INFORMATION FOR SEQ ID NO: 8:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other Nucleic Acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                      TTTGGTCCTTGTCTTATGTCCAGAATGC28                                                (2) INFORMATION FOR SEQ ID NO: 9:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other Nucleic Acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:                                      TGGAGAACACCACTTGTTGCTCCA24                                                    (2) INFORMATION FOR SEQ ID NO: 10:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other Nucleic Acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:                                     GTCTCTGAATCAGAAATCCTTCTATC26                                                  (2) INFORMATION FOR SEQ ID NO: 11:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other Nucleic Acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:                                     CATGTCAAATTTCACTGCTTCATCC25                                                   (2) INFORMATION FOR SEQ ID NO: 12:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other Nucleic Acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:                                     GCTTGCAAGCTTTATTTAGTTATGACTGATAACACTC37                                       __________________________________________________________________________

We claim:
 1. A method for selective amplification of a cDNA synthesizedfrom an RNA template in a sample consisting of a mixture of RNA anddouble-stranded DNA, which RNA and double-stranded DNA compriseconventional nucleotides and which DNA does not comprise unconventionalnucleotides, wherein the steps comprise:(a) treating said sample, in areverse transcription reaction mixture wherein said mixture comprises anunconventional nucleotide, under conditions for synthesis of an RNA:cDNAhybrid at a temperature in the range of about 55°-75° C.; (b) removingthe RNA template from said hybrid to provide single stranded cDNA; (c)treating said cDNA in an amplification reaction mixture comprising anunconventional nucleotide, to provide a double stranded primer extensionproduct DNA comprising said unconventional nucleotide, wherein saidamplification reaction mixture comprises a thermostable. DNA polymerase;(d) treating said amplification reaction mixture under conditionssufficient for denaturing said double stranded product DNA comprisingsaid unconventional nucleotide, wherein under said conditions said DNAcomprising conventional nucleotides is not denatured; and (e) repeatingsteps (c) and (d) at least once.
 2. The method of claim 1 wherein saiddouble-stranded DNA is genomic or proviral DNA.
 3. The method of claim 1wherein said denaturation conditions at step (d) comprise a reactiontemperature which is at least 10° C. less than the temperature necessaryto denature said double stranded DNA comprising conventionalnucleotides.
 4. The method of claim 1 wherein said thermostable DNApolymerase is Thermus thermophilus DNA polymerase.
 5. The method ofclaim 3 wherein said unconventional nucleotide is selected from thegroup consisting of HmdUTP and C⁷ dGTP.
 6. The method of claim 1 whereinin step (b) said removing of said RNA template comprises treating saidhybrid with an RNase activity.
 7. The method of claim 6 wherein saidRNase H activity is catalyzed by said thermostable DNA polymerase.